Down-regulating gene expression in insect plants

ABSTRACT

The present invention relates to the prevention and/or control of infestation by insect pest species. In particular, the invention relates to down-regulation of expression of target genes in insect pests using interfering ribonucleic acid (RNA) molecules. Also described are transgenic plants that (i) express or are capable of expressing interfering RNAs of the invention and (ii) are resistant to infestation by insect pest species. Compositions containing the interfering RNAs of the invention are also provided.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/869,336, filed on Jan. 12, 2018, which is a division of U.S.application Ser. No. 13/881,792, filed on Jul. 15, 2013, which is aNational Stage Entry of PCT/EP11/68910, filed Oct. 27, 2011, and whichclaims priority to U.S. Provisional Application No. 61/407,212, filedOct. 27, 2010. The disclosures of the priority applications areincorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to genetic control ofinfestation by insect pest species, particularly prevention and/orcontrol of pest infestation of plants. More specifically, the inventionrelates to down-regulation of expression of target genes in insect pestspecies by interfering ribonucleic acid (RNA) molecules. Also providedare transgenic plants that (i) express or are capable of expressinginterfering RNAs of the invention and (ii) are resistant to infestationby insect pest species. Compositions containing the interfering RNAmolecules of the invention for use in topical application onto plants orinto the environment surrounding plants are also described.

BACKGROUND TO THE INVENTION

There exists an abundance of insect pest species that can infect orinfest a wide variety of environments and host organisms. Insect pestsinclude a variety of species from the insect Orders Hemiptera (truebugs), Coleoptera (beetles), Siphonaptera (fleas), Dichyoptera(cockroaches and mantids), Lepidoptera (moths and butterflies),Orthoptera (e.g. grasshoppers) and Diptera (true flies). Pestinfestation can lead to significant damage. Insect pests that infestplant species are particularly problematic in agriculture as they cancause serious damage to crops and significantly reduce plant yields. Awide variety of different types of plant are susceptible to pestinfestation including commercial crops such as rice, cotton, soybean,potato and corn.

Traditionally, infestation with insect pests has been prevented orcontrolled through the use of chemical pesticides. However, thesechemicals are not always suitable for use in the treatment of crops asthey can be toxic to other species and can cause significantenvironmental damage. Over more recent decades, researchers havedeveloped more environmentally-friendly methods of controlling pestinfestation. For example, microorganisms such as Bacillus thuringiensisbacteria that naturally express proteins toxic to insect pests have beenused. Scientists have also isolated the genes encoding theseinsecticidal proteins and used them to generate transgenic cropsresistant to insect pests e.g. corn and cotton plants geneticallyengineered to produce proteins of the Cry family.

Although bacterial toxins have been highly successful in controllingcertain types of pest, they are not effective against all pest species.Researchers have therefore looked for other more targeted approaches topest control and in particular to RNA interference or ‘gene silencing’as a means to control pests at the genetic level.

RNA interference or ‘RNAi’ is a process whereby the expression of genesin the context of a cell or whole organism is down-regulated in asequence-specific manner. RNAi is now a well-established technique inthe art for inhibiting or down-regulating gene expression in a widevariety of organisms including pest organisms such as fungi, nematodesand insects. Furthermore, previous studies have shown thatdown-regulation of target genes in insect pest species can be used as ameans to control pest infestation.

WO2007/074405 describes methods of inhibiting expression of target genesin invertebrate pests including Colorado potato beetle. WO2005/110068describes methods of inhibiting expression of target genes ininvertebrate pests including in particular Western corn rootworm as ameans to control insect infestation. Furthermore, WO2009/091864describes compositions and methods for the suppression of target genesfrom insect pest species including pests from the Lygus genus.

Although the use of RNAi for down-regulating gene expression in pestspecies is known in the art, the success of this technique for use as apest control measure depends on selection of the most appropriate targetgenes, namely those wherein loss of function results in significantdisruption of an essential biological process and/or death of theorganism. The present invention is thus directed towards thedown-regulation of particular target genes in insect pests as a means toachieve more effective prevention and/or control of insect pestinfestation, particularly of plants.

SUMMARY OF THE INVENTION

The current inventors sought to identify improved means for preventingand/or controlling insect pest infestation using genetic approaches. Inparticular, they investigated the use of RNAi to down-regulate genes insuch a way as to impair the ability of the insect pest to survive, grow,colonize specific environments and/or infest host organisms and thuslimit the damage caused by the pest.

It has now been found by the inventors that RNAi-mediateddown-regulation of specific target genes singly or in combination withininsect pest species can be used as an effective means to control pestinfestation.

In one embodiment, the present invention relates to an interferingribonucleic acid (RNA or double stranded RNA) that inhibits ordownregulates the expression of a target gene that encodes an insectribosomal protein such as the ribosomal protein L19 (e.g. an insectorthologue of the CG2746 Dm protein), the ribosomal protein L40 (e.g. aninsect orthologue of the CG2960 Dm protein) or the ribosomal proteinS27A (e.g. an insect orthologue of the CG5271 Dm protein).

According to another embodiment the present invention relates to aninterfering ribonucleic acid (RNA or double stranded RNA) that inhibitsor downregulates the expression of a target gene that encodes an insectproteasome subunit such as the Rpn6 protein (e.g. an insect orthologueof the CG10149 Dm protein), the Pros 25 protein (e.g. an insectorthologue of the CG5266 Dm protein), the Rpn2 protein (e.g. an insectorthologue of the CG11888 Dm protein), the proteasome beta 1 subunitprotein (e.g. an insect orthologue of the CG8392 Dm protein) or the Prosbeta 2 protein (e.g. an insect orthologue of the CG3329 Dm protein).

According to still another embodiment the present invention relates toan interfering ribonucleic acid (RNA or double stranded RNA) thatinhibits or downregulates the expression of a target gene that encodesan insect β-coatomer of the COPI vesicle (e.g. an insect orthologue ofthe CG6223 Dm protein), the γ-coatomer of the COPI vesicle (e.g. aninsect orthologue of the 1528 Dm protein), the β′-coatomer protein (e.g.an insect orthologue of the CG6699 Dm protein) or the ζ-coatomer of theCOPI vesicle (e.g. an insect orthologue of the CG3948 Dm protein).

According to still another embodiment the present invention relates toan interfering ribonucleic acid (RNA or double stranded RNA) thatinhibits or downregulates the expression of a target gene that encodesan insect Tetraspanine 2 A protein which is a putative transmembranedomain protein (e.g. an insect orthologue of the CG11415 Dm protein).

According to still another embodiment the present invention relates toan interfering ribonucleic acid (RNA or double stranded RNA) thatinhibits or downregulates the expression of a target gene that encodesan insect protein belonging to the actin family (e.g. an insectorthologue of the CG5409 Dm protein) such as Actin 5C (e.g. an insectorthologue of the CG4027 Dm protein).

According to still another embodiment the present invention relates toan interfering ribonucleic acid (RNA or double stranded RNA) thatinhibits or downregulates the expression of a target gene that encodesan insect ubiquitin-5E protein (e.g. an insect orthologue of the CG32744Dm protein).

According to still another embodiment the present invention relates toan interfering ribonucleic acid (RNA or double stranded RNA) thatinhibits or downregulates the expression of a target gene that encodesan insect Sec23 protein which is a GTPase activator involved inintracellular protein transport (e.g. an insect orthologue of the CG1250Dm protein).

According to still another embodiment the present invention relates toan interfering ribonucleic acid (RNA or double stranded RNA) thatinhibits or downregulates the expression of a target gene that encodesan insect crinkled protein which is an unconventional myosin which isinvolved in motor activity (e.g. an insect orthologue of the CG7595 Dmprotein).

According to still another embodiment the present invention relates toan interfering ribonucleic acid (RNA or double stranded RNA) thatinhibits or downregulates the expression of a target gene that encodesan insect crooked neck protein which is involved in the regulation ofnuclear alternative mRNA splicing (e.g. an insect orthologue of theCG3193 Dm protein).

According to still another embodiment the present invention relates toan interfering ribonucleic acid (RNA or double stranded RNA) thatinhibits or downregulates the expression of a target gene that encodesan insect vacuolar H+-ATPase G-subunit protein (e.g. an insectorthologue of the CG6213 Dm protein).

According to still another embodiment the present invention relates toan interfering ribonucleic acid (RNA or double stranded RNA) thatinhibits or downregulates the expression of a target gene that encodesan insect Tbp-1; Tat-binding protein (e.g. an insect orthologue of theCG10370 Dm protein).

Therefore, in accordance with a first aspect of the invention, there isprovided an interfering ribonucleic acid (RNA or double stranded RNA)that functions upon uptake by an insect pest species to down-regulateexpression of a target gene in said insect pest, wherein the target gene

(i) is selected from the group of genes having a nucleotide sequencecomprising any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273,123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146, 132,133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140, 153,278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5,25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34,15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, or the complementthereof, or having a nucleotide sequence that, when the two sequencesare optimally aligned and compared, is at least 75%, preferably at least80%, 85%, 90%, 95%, 98% or 99% identical to any of SEQ ID NOs 277, 138,253, 152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144,130, 145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150,276, 137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1,21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11,31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20or 40, or the complement thereof, or

(ii) is selected from the group of genes having a nucleotide sequenceconsisting of any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273,123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146, 132,133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140, 153,278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5,25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34,15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, or the complementthereof, or

(iii) is selected from the group of genes having a nucleotide sequencecomprising a fragment of at least 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 125, 150, 175,200, 225, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900 1000,1100 or 1115 contiguous nucleotides of any of SEQ ID NOs 277, 138, 253,152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144, 130,145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150, 276,137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21,2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31,12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or40, or the complement thereof, or having a nucleotide sequence that,when said gene comprising said fragment is optimally aligned andcompared with any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273,123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146, 132,133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140, 153,278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5,25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34,15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, said nucleotidesequence is at least 75% preferably at least 80%, 85%, 90%, 95%, 98% or99% identical to any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141,273, 123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146,132, 133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140,153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24,5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14,34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, or the complementthereof, or

(iv) is selected from the group of genes having a nucleotide sequencecomprising a fragment of at least 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 125, 150, 175,200, 225, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900 1000,1100 or 1115 contiguous nucleotides of any of SEQ ID NOs 277, 138, 253,152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144, 130,145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150, 276,137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21,2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31,12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or40, or the complement thereof, and wherein when said fragment isoptimally aligned and compared with the corresponding fragment in any ofSEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273, 123, 142, 274, 124,143, 125 to 129, 144, 130, 145, 275, 131, 146, 132, 133, 147, 134, 148,135, 149, 136, 150, 276, 137, 151, 139, 140, 153, 278, 251, 254, 279,252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8,28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17,37, 18, 38, 19, 39, 20 or 40, said nucleotide sequence of said fragmentis at least 75% preferably at least 80%, 85%, 90%, 95%, 98% or 99%identical to said corresponding fragment of any of SEQ ID NOs 277, 138,253, 152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144,130, 145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150,276, 137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1,21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11,31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20or 40, or the complement thereof, or

(v) is an insect pest orthologue of a gene having a nucleotide sequencecomprising any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273,123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146, 132,133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140, 153,278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5,25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34,15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, or the complementthereof, wherein the two orthologous genes are similar in sequence tosuch a degree that when the two genes are optimally aligned andcompared, the orthologue has a sequence that is at least 75% preferablyat least 80%, 85%, 90%, 95%, 98% or 99% identical to any of thesequences represented by SEQ ID NOs 277, 138, 253, 152, 121, 122, 141,273, 123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146,132, 133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140,153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24,5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14,34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, or

(vi) is selected from the group of genes having a nucleotide sequenceencoding an amino acid sequence that, when the two amino acid sequencesare optimally aligned and compared, is at least 70% preferably at least75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the amino acid sequenceencoded by the nucleotide sequence represented in any of SEQ ID NOs 277,138, 253, 152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129,144, 130, 145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136,150, 276, 137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256,280, 1, 21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10,30, 11, 31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19,39, 20 or 40, or

(vii) is selected from the group of genes having a nucleotide sequenceencoding an amino acid sequence that, when the two amino acid sequencesare optimally aligned and compared, is at least 70% preferably at least75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the amino acid sequencerepresented in any of SEQ ID NOs 285, 242, 271, 226, 227, 281, 228, 282,229, 230 to 233, 234, 283, 235, 236, 237, 238, 239, 240, 284, 241, 243,244, 286, 269, 270, 287, 288, 206 to 225.

In a particular aspect of the invention, interfering RNA molecules ofthe current invention comprise at least one double-stranded region,typically the silencing element of the interfering RNA, comprising asense RNA strand annealed by complementary basepairing to an antisenseRNA strand wherein the sense strand of the dsRNA molecule comprises asequence of nucleotides complementary to a sequence of nucleotideslocated within the RNA transcript of the target gene.

In one embodiment, the present invention relates to an interfering RNAmolecule which comprises at least one double-stranded region, typicallythe silencing element of the interfering RNA molecule, comprising asense RNA strand annealed by complementary basepairing to an antisenseRNA strand wherein the sense strand of the dsRNA molecule comprises asequence of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 125, 150, 175, 200,225, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900 1000, 1100 or1115 contiguous nucleotides, that is at least 75% preferably at least80%, 85%, 90%, 95%, 98%, 99% or 100% complementary to a sequence ofnucleotides located within the RNA transcript of a target gene thatencodes an insect ribosomal protein such as the ribosomal protein L19(e.g. an insect orthologue of the CG2746 Dm protein), the ribosomalprotein L40 (e.g. an insect orthologue of the CG2960 Dm protein) or theribosomal protein S27A (e.g. an insect orthologue of the CG5271 Dmprotein).

According to another embodiment the present invention relates to aninterfering RNA molecule which comprises at least one double-strandedregion, typically the silencing element of the interfering RNA molecule,comprising a sense RNA strand annealed by complementary basepairing toan antisense RNA strand wherein the sense strand of the dsRNA moleculecomprises a sequence of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 125, 150,175, 200, 225, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 9001000, 1100 or 1115 contiguous nucleotides, that is at least 75%preferably at least 80%, 85%, 90%, 95%, 98%, 99% or 100% complementaryto a sequence of nucleotides located within the RNA transcript of atarget gene that encodes an insect proteasome subunit such as the Rpn6protein (e.g. an insect orthologue of the CG10149 Dm protein), the Pros25 protein (e.g. an insect orthologue of the CG5266 Dm protein), theRpn2 protein (e.g. an insect orthologue of the CG11888 Dm protein), theproteasome beta 1 subunit protein (e.g. an insect orthologue of theCG8392 Dm protein) or the Pros beta 2 protein (e.g. an insect orthologueof the CG3329 Dm protein).

According to still another embodiment the present invention relates toan interfering RNA molecule which comprises at least one double-strandedregion, typically the silencing element of the interfering RNA molecule,comprising a sense RNA strand annealed by complementary basepairing toan antisense RNA strand wherein the sense strand of the dsRNA moleculecomprises a sequence of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 125, 150,175, 200, 225, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 9001000, 1100 or 1115 contiguous nucleotides, that is at least 75%preferably at least 80%, 85%, 90%, 95%, 98%, 99% or 100% complementaryto a sequence of nucleotides located within the RNA transcript of atarget gene that encodes an insect β-coatomer of the COPI vesicle (e.g.an insect orthologue of the CG6223 Dm protein), the γ-coatomer of theCOPI vesicle (e.g. an insect orthologue of the 1528 Dm protein), theβ′-coatomer protein (e.g. an insect orthologue of the CG6699 Dm protein)or the ζ-coatomer of the COPI vesicle (e.g. an insect orthologue of theCG3948 Dm protein).

According to still another embodiment the present invention relates toan interfering RNA molecule which comprises at least one double-strandedregion, typically the silencing element of the interfering RNA molecule,comprising a sense RNA strand annealed by complementary basepairing toan antisense RNA strand wherein the sense strand of the dsRNA moleculecomprises a sequence of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 125, 150,175, 200, 225, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 9001000, 1100 or 1115 contiguous nucleotides, that is at least 75%preferably at least 80%, 85%, 90%, 95%, 98%, 99% or 100% complementaryto a sequence of nucleotides located within the RNA transcript of atarget gene that encodes an insect Tetraspanine 2 A protein which is aputative transmembrane domain protein (e.g. an insect orthologue of theCG11415 Dm protein).

According to still another embodiment the present invention relates toan interfering RNA molecule which comprises at least one double-strandedregion, typically the silencing element of the interfering RNA molecule,comprising a sense RNA strand annealed by complementary basepairing toan antisense RNA strand wherein the sense strand of the dsRNA moleculecomprises a sequence of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 125, 150,175, 200, 225, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 9001000, 1100 or 1115 contiguous nucleotides, that is at least 75%preferably at least 80%, 85%, 90%, 95%, 98%, 99% or 100% complementaryto a sequence of nucleotides located within the RNA transcript of atarget gene that encodes an insect protein belonging to the actin family(e.g. an insect orthologue of the CG5409 Dm protein) such as Actin 5C(e.g. an insect orthologue of the CG4027 Dm protein).

According to still another embodiment the present invention relates toan interfering RNA molecule which comprises at least one double-strandedregion, typically the silencing element of the interfering RNA molecule,comprising a sense RNA strand annealed by complementary basepairing toan antisense RNA strand wherein the sense strand of the dsRNA moleculecomprises a sequence of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 125, 150,175, 200, 225, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 9001000, 1100 or 1115 contiguous nucleotides, that is at least 75%preferably at least 80%, 85%, 90%, 95%, 98%, 99% or 100% complementaryto a sequence of nucleotides located within the RNA transcript of atarget gene that encodes an insect ubiquitin-5E protein (e.g. an insectorthologue of the CG32744 Dm protein).

According to still another embodiment the present invention relates toan interfering RNA molecule which comprises at least one double-strandedregion, typically the silencing element of the interfering RNA molecule,comprising a sense RNA strand annealed by complementary basepairing toan antisense RNA strand wherein the sense strand of the dsRNA moleculecomprises a sequence of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 125, 150,175, 200, 225, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 9001000, 1100 or 1115 contiguous nucleotides, that is at least 75%preferably at least 80%, 85%, 90%, 95%, 98%, 99% or 100% complementaryto a sequence of nucleotides located within the RNA transcript of atarget gene that encodes an insect Sec23 protein which is a GTPaseactivator involved in intracellular protein transport (e.g. an insectorthologue of the CG1250 Dm protein).

According to still another embodiment the present invention relates toan interfering RNA molecule which comprises at least one double-strandedregion, typically the silencing element of the interfering RNA molecule,comprising a sense RNA strand annealed by complementary basepairing toan antisense RNA strand wherein the sense strand of the dsRNA moleculecomprises a sequence of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 125, 150,175, 200, 225, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 9001000, 1100 or 1115 contiguous nucleotides, that is at least 75%preferably at least 80%, 85%, 90%, 95%, 98%, 99% or 100% complementaryto a sequence of nucleotides located within the RNA transcript of atarget gene that encodes an insect crinkled protein which is anunconventional myosin which is involved in motor activity (e.g. aninsect orthologue of the CG7595 Dm protein).

According to still another embodiment the present invention relates toan interfering RNA molecule which comprises at least one double-strandedregion, typically the silencing element of the interfering RNA molecule,comprising a sense RNA strand annealed by complementary basepairing toan antisense RNA strand wherein the sense strand of the dsRNA moleculecomprises a sequence of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 125, 150,175, 200, 225, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 9001000, 1100 or 1115 contiguous nucleotides, that is at least 75%preferably at least 80%, 85%, 90%, 95%, 98%, 99% or 100% complementaryto a sequence of nucleotides located within the RNA transcript of atarget gene that encodes an insect crooked neck protein which isinvolved in the regulation of nuclear alternative mRNA splicing (e.g. aninsect orthologue of the CG3193 Dm protein).

According to still another embodiment the present invention relates toan interfering RNA molecule which comprises at least one double-strandedregion, typically the silencing element of the interfering RNA molecule,comprising a sense RNA strand annealed by complementary basepairing toan antisense RNA strand wherein the sense strand of the dsRNA moleculecomprises a sequence of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 125, 150,175, 200, 225, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 9001000, 1100 or 1115 contiguous nucleotides, that is at least 75%preferably at least 80%, 85%, 90%, 95%, 98%, 99% or 100% complementaryto a sequence of nucleotides located within the RNA transcript of atarget gene that encodes an insect vacuolar H+-ATPase G-subunit protein(e.g. an insect orthologue of the CG6213 Dm protein).

According to still another embodiment the present invention relates toan interfering RNA molecule which comprises at least one double-strandedregion, typically the silencing element of the interfering RNA molecule,comprising a sense RNA strand annealed by complementary basepairing toan antisense RNA strand wherein the sense strand of the dsRNA moleculecomprises a sequence of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 125, 150,175, 200, 225, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 9001000, 1100 or 1115 contiguous nucleotides, that is at least 75%preferably at least 80%, 85%, 90%, 95%, 98%, 99% or 100% complementaryto a sequence of nucleotides located within the RNA transcript of atarget gene that encodes an insect Tbp-1; Tat-binding protein (e.g. aninsect orthologue of the CG10370 Dm protein).

In accordance with a second aspect of the invention, there is providedan isolated polynucleotide selected from the group consisting of:

(i) a polynucleotide which comprises at least 21, preferably at least22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90,100, 110, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450, 500, 550,600, 700, 800, 900, 1000, 1100 or 1115 contiguous nucleotides of anucleotide sequence as represented by any of SEQ ID NOs 277, 138, 253,152, 198 to 201, 121, 122, 141, 154 to 157, 273, 123, 142, 158 to 161,274, 124, 143, 162 to 165, 125 to 129, 144, 166 to 169, 130, 145, 170 to173, 275, 131, 146, 174 to 177, 132, 133, 147, 178 to 181, 134, 148, 182to 185, 135, 149, 186 to 189, 136, 150, 190 to 193, 276, 137, 151, 194to 197, 139, 140, 153, 202 to 205, 278, 251, 254, 257 to 260, 279, 252,255, 256, 261 to 268, 280, 1, 21, 41 to 44, 2, 22, 45 to 48, 3, 23, 49to 52, 4, 24, 53 to 56, 5, 25, 57 to 60, 6, 26, 61 to 64, 7, 27, 65 to68, 8, 28, 69 to 72, 9, 29, 73 to 76, 10, 30, 77 to 80, 11, 31, 81 to84, 12, 32, 85 to 88, 13, 33, 89 to 92, 14, 34, 93 to 96, 15, 35, 97 to100, 16, 36, 101 to 104, 17, 37, 105 to 108, 18, 38, 109 to 112, 19, 39,113 to 116, 20, 40, 117 to 120, or the complement thereof, or

(ii) a polynucleotide which consists of at least 21, preferably at least22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90,100, 110, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450, 500, 550,600, 700, 800, 900, 1000, 1100 or 1115 contiguous nucleotides of anucleotide sequence as represented by any of SEQ ID NOs 277, 138, 253,152, 198 to 201, 121, 122, 141, 154 to 157, 273, 123, 142, 158 to 161,274, 124, 143, 162 to 165, 125 to 129, 144, 166 to 169, 130, 145, 170 to173, 275, 131, 146, 174 to 177, 132, 133, 147, 178 to 181, 134, 148, 182to 185, 135, 149, 186 to 189, 136, 150, 190 to 193, 276, 137, 151, 194to 197, 139, 140, 153, 202 to 205, 278, 251, 254, 257 to 260, 279, 252,255, 256, 261 to 268, 280, 1, 21, 41 to 44, 2, 22, 45 to 48, 3, 23, 49to 52, 4, 24, 53 to 56, 5, 25, 57 to 60, 6, 26, 61 to 64, 7, 27, 65 to68, 8, 28, 69 to 72, 9, 29, 73 to 76, 10, 30, 77 to 80, 11, 31, 81 to84, 12, 32, 85 to 88, 13, 33, 89 to 92, 14, 34, 93 to 96, 15, 35, 97 to100, 16, 36, 101 to 104, 17, 37, 105 to 108, 18, 38, 109 to 112, 19, 39,113 to 116, 20, 40, 117 to 120, or the complement thereof, or

(iii) a polynucleotide which comprises at least 21, preferably at least22, 23 or 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80,90, 100, 110, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450, 500,550, 600, 700, 800, 900, 1000, 1100 or 1115 contiguous nucleotides of anucleotide sequence as represented in any of SEQ ID NOs 277, 138, 253,152, 198 to 201, 121, 122, 141, 154 to 157, 273, 123, 142, 158 to 161,274, 124, 143, 162 to 165, 125 to 129, 144, 166 to 169, 130, 145, 170 to173, 275, 131, 146, 174 to 177, 132, 133, 147, 178 to 181, 134, 148, 182to 185, 135, 149, 186 to 189, 136, 150, 190 to 193, 276, 137, 151, 194to 197, 139, 140, 153, 202 to 205, 278, 251, 254, 257 to 260, 279, 252,255, 256, 261 to 268, 280, 1, 21, 41 to 44, 2, 22, 45 to 48, 3, 23, 49to 52, 4, 24, 53 to 56, 5, 25, 57 to 60, 6, 26, 61 to 64, 7, 27, 65 to68, 8, 28, 69 to 72, 9, 29, 73 to 76, 10, 30, 77 to 80, 11, 31, 81 to84, 12, 32, 85 to 88, 13, 33, 89 to 92, 14, 34, 93 to 96, 15, 35, 97 to100, 16, 36, 101 to 104, 17, 37, 105 to 108, 18, 38, 109 to 112, 19, 39,113 to 116, 20, 40, 117 to 120, or the complement thereof, that, whenthe two sequences are optimally aligned and compared, saidpolynucleotide is at least 75% preferably at least 80%, 85%, 90%, 95%,98% or 99% identical to any of SEQ ID NOs 277, 138, 253, 152, 198 to201, 121, 122, 141, 154 to 157, 273, 123, 142, 158 to 161, 274, 124,143, 162 to 165, 125 to 129, 144, 166 to 169, 130, 145, 170 to 173, 275,131, 146, 174 to 177, 132, 133, 147, 178 to 181, 134, 148, 182 to 185,135, 149, 186 to 189, 136, 150, 190 to 193, 276, 137, 151, 194 to 197,139, 140, 153, 202 to 205, 278, 251, 254, 257 to 260, 279, 252, 255,256, 261 to 268, 280, 1, 21, 41 to 44, 2, 22, 45 to 48, 3, 23, 49 to 52,4, 24, 53 to 56, 5, 25, 57 to 60, 6, 26, 61 to 64, 7, 27, 65 to 68, 8,28, 69 to 72, 9, 29, 73 to 76, 10, 30, 77 to 80, 11, 31, 81 to 84, 12,32, 85 to 88, 13, 33, 89 to 92, 14, 34, 93 to 96, 15, 35, 97 to 100, 16,36, 101 to 104, 17, 37, 105 to 108, 18, 38, 109 to 112, 19, 39, 113 to116, 20, 40, 117 to 120, or the complement thereof, or

(iv) a polynucleotide which comprises a fragment of at least 21,preferably at least 22, 23 or 24, 25, 26, 27, 28, 29, 30, 35, 40, 45,50, 55, 60, 70, 80, 90, 100, 110, 125, 150, 175, 200, 225, 250, 300,350, 400, 450, 500, 550, 600, 700, 800, 900, 1000, 1100 or 1115contiguous nucleotides of a nucleotide as represented in any of SEQ IDNOs 277, 138, 253, 152, 198 to 201, 121, 122, 141, 154 to 157, 273, 123,142, 158 to 161, 274, 124, 143, 162 to 165, 125 to 129, 144, 166 to 169,130, 145, 170 to 173, 275, 131, 146, 174 to 177, 132, 133, 147, 178 to181, 134, 148, 182 to 185, 135, 149, 186 to 189, 136, 150, 190 to 193,276, 137, 151, 194 to 197, 139, 140, 153, 202 to 205, 278, 251, 254, 257to 260, 279, 252, 255, 256, 261 to 268, 280, 1, 21, 41 to 44, 2, 22, 45to 48, 3, 23, 49 to 52, 4, 24, 53 to 56, 5, 25, 57 to 60, 6, 26, 61 to64, 7, 27, 65 to 68, 8, 28, 69 to 72, 9, 29, 73 to 76, 10, 30, 77 to 80,11, 31, 81 to 84, 12, 32, 85 to 88, 13, 33, 89 to 92, 14, 34, 93 to 96,15, 35, 97 to 100, 16, 36, 101 to 104, 17, 37, 105 to 108, 18, 38, 109to 112, 19, 39, 113 to 116, 20, 40, 117 to 120, or the complementthereof, and wherein said fragment or said complement has a nucleotidesequence that, when said fragment is optimally aligned and compared withthe corresponding fragment in any of SEQ ID NOs 277, 138, 253, 152, 198to 201, 121, 122, 141, 154 to 157, 273, 123, 142, 158 to 161, 274, 124,143, 162 to 165, 125 to 129, 144, 166 to 169, 130, 145, 170 to 173, 275,131, 146, 174 to 177, 132, 133, 147, 178 to 181, 134, 148, 182 to 185,135, 149, 186 to 189, 136, 150, 190 to 193, 276, 137, 151, 194 to 197,139, 140, 153, 202 to 205, 278, 251, 254, 257 to 260, 279, 252, 255,256, 261 to 268, 280, 1, 21, 41 to 44, 2, 22, 45 to 48, 3, 23, 49 to 52,4, 24, 53 to 56, 5, 25, 57 to 60, 6, 26, 61 to 64, 7, 27, 65 to 68, 8,28, 69 to 72, 9, 29, 73 to 76, 10, 30, 77 to 80, 11, 31, 81 to 84, 12,32, 85 to 88, 13, 33, 89 to 92, 14, 34, 93 to 96, 15, 35, 97 to 100, 16,36, 101 to 104, 17, 37, 105 to 108, 18, 38, 109 to 112, 19, 39, 113 to116, 20, 40, 117 to 120, said nucleotide sequence is at least 75%preferably at least 80%, 85%, 90%, 95%, 98% or 99% identical to saidcorresponding fragment of any of SEQ ID NOs 277, 138, 253, 152, 198 to201, 121, 122, 141, 154 to 157, 273, 123, 142, 158 to 161, 274, 124,143, 162 to 165, 125 to 129, 144, 166 to 169, 130, 145, 170 to 173, 275,131, 146, 174 to 177, 132, 133, 147, 178 to 181, 134, 148, 182 to 185,135, 149, 186 to 189, 136, 150, 190 to 193, 276, 137, 151, 194 to 197,139, 140, 153, 202 to 205, 278, 251, 254, 257 to 260, 279, 252, 255,256, 261 to 268, 280, 1, 21, 41 to 44, 2, 22, 45 to 48, 3, 23, 49 to 52,4, 24, 53 to 56, 5, 25, 57 to 60, 6, 26, 61 to 64, 7, 27, 65 to 68, 8,28, 69 to 72, 9, 29, 73 to 76, 10, 30, 77 to 80, 11, 31, 81 to 84, 12,32, 85 to 88, 13, 33, 89 to 92, 14, 34, 93 to 96, 15, 35, 97 to 100, 16,36, 101 to 104, 17, 37, 105 to 108, 18, 38, 109 to 112, 19, 39, 113 to116, 20, 40, 117 to 120 or the complement thereof, or

(v) a polynucleotide which consists of a fragment of at least 21,preferably at least 22, 23 or 24, 25, 26, 27, 28, 29, 30, 35, 40, 45,50, 55, 60, 70, 80, 90, 100, 110, 125, 150, 175, 200, 225, 250, 300,350, 400, 450, 500, 550, 600, 700, 800, 900, 1000, 1100 or 1115contiguous nucleotides of a nucleotide as represented in any of SEQ IDNOs 277, 138, 253, 152, 198 to 201, 121, 122, 141, 154 to 157, 273, 123,142, 158 to 161, 274, 124, 143, 162 to 165, 125 to 129, 144, 166 to 169,130, 145, 170 to 173, 275, 131, 146, 174 to 177, 132, 133, 147, 178 to181, 134, 148, 182 to 185, 135, 149, 186 to 189, 136, 150, 190 to 193,276, 137, 151, 194 to 197, 139, 140, 153, 202 to 205, 278, 251, 254, 257to 260, 279, 252, 255, 256, 261 to 268, 280, 1, 21, 41 to 44, 2, 22, 45to 48, 3, 23, 49 to 52, 4, 24, 53 to 56, 5, 25, 57 to 60, 6, 26, 61 to64, 7, 27, 65 to 68, 8, 28, 69 to 72, 9, 29, 73 to 76, 10, 30, 77 to 80,11, 31, 81 to 84, 12, 32, 85 to 88, 13, 33, 89 to 92, 14, 34, 93 to 96,15, 35, 97 to 100, 16, 36, 101 to 104, 17, 37, 105 to 108, 18, 38, 109to 112, 19, 39, 113 to 116, 20, 40, 117 to 120, or the complementthereof, and wherein said fragment or said complement has a nucleotidesequence that, when said fragment is optimally aligned and compared withthe corresponding fragment in any of SEQ ID NOs 277, 138, 253, 152, 198to 201, 121, 122, 141, 154 to 157, 273, 123, 142, 158 to 161, 274, 124,143, 162 to 165, 125 to 129, 144, 166 to 169, 130, 145, 170 to 173, 275,131, 146, 174 to 177, 132, 133, 147, 178 to 181, 134, 148, 182 to 185,135, 149, 186 to 189, 136, 150, 190 to 193, 276, 137, 151, 194 to 197,139, 140, 153, 202 to 205, 278, 251, 254, 257 to 260, 279, 252, 255,256, 261 to 268, 280, 1, 21, 41 to 44, 2, 22, 45 to 48, 3, 23, 49 to 52,4, 24, 53 to 56, 5, 25, 57 to 60, 6, 26, 61 to 64, 7, 27, 65 to 68, 8,28, 69 to 72, 9, 29, 73 to 76, 10, 30, 77 to 80, 11, 31, 81 to 84, 12,32, 85 to 88, 13, 33, 89 to 92, 14, 34, 93 to 96, 15, 35, 97 to 100, 16,36, 101 to 104, 17, 37, 105 to 108, 18, 38, 109 to 112, 19, 39, 113 to116, 20, 40, 117 to 120, said nucleotide sequence is at least 75%preferably at least 80%, 85%, 90%, 95%, 98% or 99% identical to saidcorresponding fragment of any of SEQ ID NOs 277, 138, 253, 152, 198 to201, 121, 122, 141, 154 to 157, 273, 123, 142, 158 to 161, 274, 124,143, 162 to 165, 125 to 129, 144, 166 to 169, 130, 145, 170 to 173, 275,131, 146, 174 to 177, 132, 133, 147, 178 to 181, 134, 148, 182 to 185,135, 149, 186 to 189, 136, 150, 190 to 193, 276, 137, 151, 194 to 197,139, 140, 153, 202 to 205, 278, 251, 254, 257 to 260, 279, 252, 255,256, 261 to 268, 280, 1, 21, 41 to 44, 2, 22, 45 to 48, 3, 23, 49 to 52,4, 24, 53 to 56, 5, 25, 57 to 60, 6, 26, 61 to 64, 7, 27, 65 to 68, 8,28, 69 to 72, 9, 29, 73 to 76, 10, 30, 77 to 80, 11, 31, 81 to 84, 12,32, 85 to 88, 13, 33, 89 to 92, 14, 34, 93 to 96, 15, 35, 97 to 100, 16,36, 101 to 104, 17, 37, 105 to 108, 18, 38, 109 to 112, 19, 39, 113 to116, 20, 40, 117 to 120 or the complement thereof, or

(vi) a polynucleotide encoding an amino acid sequence that, when the twoamino acid sequences are optimally aligned and compared, is at least 70%preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to theamino acid sequence encoded by the nucleotide sequence represented inany of SEQ ID NOs 277, 138, 253, 152, 198 to 201, 121, 122, 141, 154 to157, 273, 123, 142, 158 to 161, 274, 124, 143, 162 to 165, 125 to 129,144, 166 to 169, 130, 145, 170 to 173, 275, 131, 146, 174 to 177, 132,133, 147, 178 to 181, 134, 148, 182 to 185, 135, 149, 186 to 189, 136,150, 190 to 193, 276, 137, 151, 194 to 197, 139, 140, 153, 202 to 205,278, 251, 254, 257 to 260, 279, 252, 255, 256, 261 to 268, 280, 1, 21,41 to 44, 2, 22, 45 to 48, 3, 23, 49 to 52, 4, 24, 53 to 56, 5, 25, 57to 60, 6, 26, 61 to 64, 7, 27, 65 to 68, 8, 28, 69 to 72, 9, 29, 73 to76, 10, 30, 77 to 80, 11, 31, 81 to 84, 12, 32, 85 to 88, 13, 33, 89 to92, 14, 34, 93 to 96, 15, 35, 97 to 100, 16, 36, 101 to 104, 17, 37, 105to 108, 18, 38, 109 to 112, 19, 39, 113 to 116, 20, 40, 117 to 120, or

(vii) a polynucleotide encoding an amino acid sequence that, when thetwo amino acid sequences are optimally aligned and compared, is at least70% preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical tothe amino acid sequence represented in any of SEQ ID NOs 285, 242, 271,226, 227, 281, 228, 282, 229, 230 to 233, 234, 283, 235, 236, 237, 238,239, 240, 284, 241, 243, 244, 286, 269, 270, 287, 288, 206 to 225,

and

wherein said polynucleotide is no longer than 10000, 9000, 8000, 7000,6000, 5000, 4000, 3000, 2000 or 1500 nucleotides.

In a particular aspect of the invention, the isolated polynucleotide ispart of an interfering RNA molecule, typically part of the silencingelement, comprising at least one double-stranded region comprising asense RNA strand annealed by complementary basepairing to an antisenseRNA strand wherein the sense strand of the dsRNA molecule comprises asequence of nucleotides complementary to a sequence of nucleotideslocated within the RNA transcript of the target gene. More particularly,the isolated polynucleotide is cloned in a DNA construct in a sense andantisense orientation so that upon transcription of the sense andantisense polynucleotide a dsRNA molecule is formed, which functionsupon uptake by a pest to inhibit or down-regulate the expression of atarget gene within said pest.

In one embodiment, the present invention relates to an isolatedpolynucleotide that is cloned in a DNA construct in a sense andantisense orientation so that upon transcription of the sense andantisense polynucleotide a dsRNA molecule is formed, which functionsupon uptake by an insect to inhibit or down-regulate the expression of atarget gene that encodes an insect ribosomal protein such as theribosomal protein L19 (e.g. an insect orthologue of the CG2746 Dmprotein), the ribosomal protein L40 (e.g. an insect orthologue of theCG2960 Dm protein) or the ribosomal protein S27A (e.g. an insectorthologue of the CG5271 Dm protein).

According to another embodiment the present invention relates to anisolated polynucleotide that is cloned in a DNA construct in a sense andantisense orientation so that upon transcription of the sense andantisense polynucleotide a dsRNA molecule is formed, which functionsupon uptake by an insect to inhibit or down-regulate the expression of atarget gene that encodes an insect proteasome subunit such as the Rpn6protein (e.g. an insect orthologue of the CG10149 Dm protein), the Pros25 protein (e.g. an insect orthologue of the CG5266 Dm protein), theRpn2 protein (e.g. an insect orthologue of the CG11888 Dm protein), theproteasome beta 1 subunit protein (e.g. an insect orthologue of theCG8392 Dm protein) or the Pros beta 2 protein (e.g. an insect orthologueof the CG3329 Dm protein).

According to still another embodiment the present invention relates toan isolated polynucleotide that is cloned in a DNA construct in a senseand antisense orientation so that upon transcription of the sense andantisense polynucleotide a dsRNA molecule is formed, which functionsupon uptake by an insect to inhibit or down-regulate the expression of atarget gene that encodes an insect β-coatomer of the COPI vesicle (e.g.an insect orthologue of the CG6223 Dm protein), the γ-coatomer of theCOPI vesicle (e.g. an insect orthologue of the 1528 Dm protein), theβ′-coatomer protein (e.g. an insect orthologue of the CG6699 Dm protein)or the ζ-coatomer of the COPI vesicle (e.g. an insect orthologue of theCG3948 Dm protein).

According to still another embodiment the present invention relates toan isolated polynucleotide that is cloned in a DNA construct in a senseand antisense orientation so that upon transcription of the sense andantisense polynucleotide a dsRNA molecule is formed, which functionsupon uptake by an insect to inhibit or down-regulate the expression of atarget gene that encodes an insect Tetraspanine 2 A protein which is aputative transmembrane domain protein (e.g. an insect orthologue of theCG11415 Dm protein).

According to still another embodiment the present invention relates toan isolated polynucleotide that is cloned in a DNA construct in a senseand antisense orientation so that upon transcription of the sense andantisense polynucleotide a dsRNA molecule is formed, which functionsupon uptake by an insect to inhibit or down-regulate the expression of atarget gene that encodes an insect protein belonging to the actin family(e.g. an insect orthologue of the CG5409 Dm protein) such as Actin 5C(e.g. an insect orthologue of the CG4027 Dm protein).

According to still another embodiment the present invention relates toan isolated polynucleotide that is cloned in a DNA construct in a senseand antisense orientation so that upon transcription of the sense andantisense polynucleotide a dsRNA molecule is formed, which functionsupon uptake by an insect to inhibit or down-regulate the expression of atarget gene that encodes an insect ubiquitin-5E protein (e.g. an insectorthologue of the CG32744 Dm protein).

According to still another embodiment the present invention relates toan isolated polynucleotide that is cloned in a DNA construct in a senseand antisense orientation so that upon transcription of the sense andantisense polynucleotide a dsRNA molecule is formed, which functionsupon uptake by an insect to inhibit or down-regulate the expression of atarget gene that encodes an insect Sec23 protein which is a GTPaseactivator involved in intracellular protein transport (e.g. an insectorthologue of the CG1250 Dm protein).

According to still another embodiment the present invention relates toan isolated polynucleotide that is cloned in a DNA construct in a senseand antisense orientation so that upon transcription of the sense andantisense polynucleotide a dsRNA molecule is formed, which functionsupon uptake by an insect to inhibit or down-regulate the expression of atarget gene that encodes an insect crinkled protein which is anunconventional myosin which is involved in motor activity (e.g. aninsect orthologue of the CG7595 Dm protein).

According to still another embodiment the present invention relates toan isolated polynucleotide that is cloned in a DNA construct in a senseand antisense orientation so that upon transcription of the sense andantisense polynucleotide a dsRNA molecule is formed, which functionsupon uptake by an insect to inhibit or down-regulate the expression of atarget gene that encodes an insect crooked neck protein which isinvolved in the regulation of nuclear alternative mRNA splicing (e.g. aninsect orthologue of the CG3193 Dm protein).

According to still another embodiment the present invention relates toan isolated polynucleotide that is cloned in a DNA construct in a senseand antisense orientation so that upon transcription of the sense andantisense polynucleotide a dsRNA molecule is formed, which functionsupon uptake by an insect to inhibit or down-regulate the expression of atarget gene that encodes an insect vacuolar H+-ATPase G-subunit protein(e.g. an insect orthologue of the CG6213 Dm protein).

According to still another embodiment the present invention relates toan isolated polynucleotide that is cloned in a DNA construct in a senseand antisense orientation so that upon transcription of the sense andantisense polynucleotide a dsRNA molecule is formed, which functionsupon uptake by an insect to inhibit or down-regulate the expression of atarget gene that encodes an insect Tbp-1; Tat-binding protein (e.g. aninsect orthologue of the CG10370 Dm protein).

In accordance with a third aspect of the invention there is provided acomposition for preventing and/or controlling insect pest infestationcomprising at least one interfering ribonucleic acid (RNA) and at leastone suitable carrier, excipient or diluent, wherein the interfering RNAfunctions upon uptake by the pest to down-regulate the expression of atarget gene within said pest, wherein the target gene

(i) is selected from the group of genes having a nucleotide sequencecomprising any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273,123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146, 132,133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140, 153,278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5,25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34,15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, or the complementthereof, or having a nucleotide sequence that, when the two sequencesare optimally aligned and compared, is at least 75%, preferably at least80%, 85%, 90%, 95%, 98% or 99% identical to any of SEQ ID NOs 277, 138,253, 152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144,130, 145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150,276, 137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1,21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11,31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20or 40, or the complement thereof, or

(ii) is selected from the group of genes having a nucleotide sequenceconsisting of any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273,123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146, 132,133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140, 153,278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5,25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34,15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, or the complementthereof, or

(iii) is selected from the group of genes having a nucleotide sequencecomprising a fragment of at least 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 125, 150, 175,200, 225, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900 1000,1100 or 1115 contiguous nucleotides of any of SEQ ID NOs 277, 138, 253,152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144, 130,145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150, 276,137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21,2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31,12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or40, or the complement thereof, or having a nucleotide sequence that,when said gene comprising said fragment is optimally aligned andcompared with any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273,123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146, 132,133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140, 153,278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5,25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34,15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, said nucleotidesequence is at least 75% preferably at least 80%, 85%, 90%, 95%, 98% or99% identical to any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141,273, 123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146,132, 133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140,153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24,5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14,34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, or the complementthereof, or

(iv) is selected from the group of genes having a nucleotide sequencecomprising a fragment of at least 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 125, 150, 175,200, 225, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900 1000,1100 or 1115 contiguous nucleotides of any of SEQ ID NOs 277, 138, 253,152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144, 130,145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150, 276,137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21,2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31,12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or40, or the complement thereof, and wherein when said fragment isoptimally aligned and compared with the corresponding fragment in any ofSEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273, 123, 142, 274, 124,143, 125 to 129, 144, 130, 145, 275, 131, 146, 132, 133, 147, 134, 148,135, 149, 136, 150, 276, 137, 151, 139, 140, 153, 278, 251, 254, 279,252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8,28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17,37, 18, 38, 19, 39, 20 or 40, said nucleotide sequence of said fragmentis at least 75% preferably at least 80%, 85%, 90%, 95%, 98% or 99%identical to said corresponding fragment of any of SEQ ID NOs 277, 138,253, 152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144,130, 145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150,276, 137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1,21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11,31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20or 40, or the complement thereof, or

(v) is an insect pest orthologue of a gene having a nucleotide sequencecomprising any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273,123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146, 132,133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140, 153,278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5,25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34,15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, or the complementthereof, wherein the two orthologous genes are similar in sequence tosuch a degree that when the two genes are optimally aligned andcompared, the orthologue has a sequence that is at least 75% preferablyat least 80%, 85%, 90%, 95%, 98% or 99% identical to any of thesequences represented by SEQ ID NOs 277, 138, 253, 152, 121, 122, 141,273, 123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146,132, 133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140,153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24,5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14,34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, or

(vi) is selected from the group of genes having a nucleotide sequenceencoding an amino acid sequence that, when the two amino acid sequencesare optimally aligned and compared, is at least 70% preferably at least75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the amino acid sequenceencoded by the nucleotide sequence represented in any of SEQ ID NOs 277,138, 253, 152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129,144, 130, 145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136,150, 276, 137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256,280, 1, 21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10,30, 11, 31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19,39, 20 or 40, or

(vii) is selected from the group of genes having a nucleotide sequenceencoding an amino acid sequence that, when the two amino acid sequencesare optimally aligned and compared, is at least 70% preferably at least75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the amino acid sequencerepresented in any of SEQ ID NOs 285, 242, 271, 226, 227, 281, 228, 282,229, 230 to 233, 234, 283, 235, 236, 237, 238, 239, 240, 284, 241, 243,244, 286, 269, 270, 287, 288, 206 to 225.

In accordance with a fourth aspect of the invention, there is provided amethod for down-regulating expression of a target gene in an insect pestspecies comprising contacting said insect pest species with an effectiveamount of at least one interfering ribonucleic acid (RNA) or aneffective amount of a composition comprising at least one interferingribonucleic acid (RNA) and at least one suitable carrier, excipient ordiluent, wherein the interfering RNA functions upon uptake by the pestto down-regulate the expression of a target gene within said pest, andwherein the target gene

(i) is selected from the group of genes having a nucleotide sequencecomprising any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273,123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146, 132,133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140, 153,278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5,25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34,15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, or the complementthereof, or having a nucleotide sequence that, when the two sequencesare optimally aligned and compared, is at least 75%, preferably at least80%, 85%, 90%, 95%, 98% or 99% identical any of SEQ ID NOs 277, 138,253, 152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144,130, 145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150,276, 137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1,21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11,31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20or 40, or the complement thereof, or

(ii) is selected from the group of genes having a nucleotide sequenceconsisting of any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273,123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146, 132,133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140, 153,278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5,25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34,15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, or the complementthereof, or

(iii) is selected from the group of genes having a nucleotide sequencecomprising a fragment of at least 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 125, 150, 175,200, 225, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900 1000,1100 or 1115 contiguous nucleotides of any of SEQ ID NOs 277, 138, 253,152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144, 130,145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150, 276,137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21,2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31,12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or40, or the complement thereof, or having a nucleotide sequence that,when said gene comprising said fragment is optimally aligned andcompared with any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273,123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146, 132,133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140, 153,278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5,25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34,15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, said nucleotidesequence is at least 75% preferably at least 80%, 85%, 90%, 95%, 98% or99% identical to any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141,273, 123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146,132, 133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140,153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24,5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14,34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, or the complementthereof, or

(iv) is selected from the group of genes having a nucleotide sequencecomprising a fragment of at least 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 125, 150, 175,200, 225, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900 1000,1100 or 1115 contiguous nucleotides of any of SEQ ID NOs 277, 138, 253,152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144, 130,145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150, 276,137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21,2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31,12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or40, or the complement thereof, and wherein when said fragment isoptimally aligned and compared with the corresponding fragment in any ofSEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273, 123, 142, 274, 124,143, 125 to 129, 144, 130, 145, 275, 131, 146, 132, 133, 147, 134, 148,135, 149, 136, 150, 276, 137, 151, 139, 140, 153, 278, 251, 254, 279,252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8,28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17,37, 18, 38, 19, 39, 20 or 40, said nucleotide sequence of said fragmentis at least 75% preferably at least 80%, 85%, 90%, 95%, 98% or 99%identical to said corresponding fragment of any of SEQ ID NOs 277, 138,253, 152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144,130, 145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150,276, 137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1,21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11,31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20or 40, or the complement thereof, or

(v) is an insect pest orthologue of a gene having a nucleotide sequencecomprising any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273,123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146, 132,133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140, 153,278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5,25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34,15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, or the complementthereof, wherein the two orthologous genes are similar in sequence tosuch a degree that when the two genes are optimally aligned andcompared, the orthologue has a sequence that is at least 75% preferablyat least 80%, 85%, 90%, 95%, 98% or 99% identical to any of thesequences represented by SEQ ID NOs 277, 138, 253, 152, 121, 122, 141,273, 123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146,132, 133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140,153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24,5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14,34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, or

(vi) is selected from the group of genes having a nucleotide sequenceencoding an amino acid sequence that, when the two amino acid sequencesare optimally aligned and compared, is at least 70% preferably at least75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the amino acid sequenceencoded by the nucleotide sequence represented in any of SEQ ID NOs 277,138, 253, 152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129,144, 130, 145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136,150, 276, 137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256,280, 1, 21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10,30, 11, 31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19,39, 20 or 40, or

(vii) is selected from the group of genes having a nucleotide sequenceencoding an amino acid sequence that, when the two amino acid sequencesare optimally aligned and compared, is at least 70% preferably at least75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the amino acid sequencerepresented in any of SEQ ID NOs 285, 242, 271, 226, 227, 281, 228, 282,229, 230 to 233, 234, 283, 235, 236, 237, 238, 239, 240, 284, 241, 243,244, 286, 269, 270, 287, 288, 206 to 225.

Preferably, the method of the invention finds practical application inthe prevention and/or control of insect pest infestation, in particular,control of pest infestation of crop plants such as but not limited torice, cotton, strawberries, seed crops such as alfalfa, soy, potato,tomato, canola, sunflower, sorghum, pearl millet, corn, eggplant, pepperand tobacco. In addition, the interfering RNA of the invention may beintroduced into the plants to be protected by routine geneticengineering techniques.

Therefore, in accordance with a fifth aspect of the invention, there isprovided a method for generating a transgenic plant resistant toinfestation by an insect pest species comprising:

(a) transforming a plant cell with a DNA construct comprising apolynucleotide sequence encoding an interfering ribonucleic acid (RNA)that functions upon uptake by an insect pest species to down-regulateexpression of a target gene in said insect pest species, wherein thetarget gene

(i) is selected from the group of genes having a nucleotide sequencecomprising any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273,123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146, 132,133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140, 153,278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5,25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34,15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, or the complementthereof, or having a nucleotide sequence that, when the two sequencesare optimally aligned and compared, is at least 75%, preferably at least80%, 85%, 90%, 95%, 98% or 99% identical any of SEQ ID NOs 277, 138,253, 152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144,130, 145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150,276, 137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1,21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11,31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20or 40, or the complement thereof, or

(ii) is selected from the group of genes having a nucleotide sequenceconsisting of any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273,123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146, 132,133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140, 153,278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5,25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34,15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, or the complementthereof, or

(iii) is selected from the group of genes having a nucleotide sequencecomprising a fragment of at least 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 125, 150, 175,200, 225, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900 1000,1100 or 1115 contiguous nucleotides of any of SEQ ID NOs 277, 138, 253,152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144, 130,145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150, 276,137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21,2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31,12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or40, or the complement thereof, or having a nucleotide sequence that,when said gene comprising said fragment is optimally aligned andcompared with any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273,123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146, 132,133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140, 153,278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5,25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34,15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, said nucleotidesequence is at least 75% preferably at least 80%, 85%, 90%, 95%, 98% or99% identical to any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141,273, 123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146,132, 133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140,153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24,5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14,34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, or the complementthereof, or

(iv) is selected from the group of genes having a nucleotide sequencecomprising a fragment of at least 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 125, 150, 175,200, 225, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900 1000,1100 or 1115 contiguous nucleotides of any of SEQ ID NOs 277, 138, 253,152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144, 130,145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150, 276,137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21,2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31,12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or40, or the complement thereof, and wherein when said fragment isoptimally aligned and compared with the corresponding fragment in any ofSEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273, 123, 142, 274, 124,143, 125 to 129, 144, 130, 145, 275, 131, 146, 132, 133, 147, 134, 148,135, 149, 136, 150, 276, 137, 151, 139, 140, 153, 278, 251, 254, 279,252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8,28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17,37, 18, 38, 19, 39, 20 or 40, said nucleotide sequence of said fragmentis at least 75% preferably at least 80%, 85%, 90%, 95%, 98% or 99%identical to said corresponding fragment of any of SEQ ID NOs 277, 138,253, 152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144,130, 145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150,276, 137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1,21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11,31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20or 40, or the complement thereof, or

(v) is an insect pest orthologue of a gene having a nucleotide sequencecomprising any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273,123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146, 132,133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140, 153,278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5,25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34,15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, or the complementthereof, wherein the two orthologous genes are similar in sequence tosuch a degree that when the two genes are optimally aligned andcompared, the orthologue has a sequence that is at least 75% preferablyat least 80%, 85%, 90%, 95%, 98% or 99% identical to any of thesequences represented by SEQ ID NOs 277, 138, 253, 152, 121, 122, 141,273, 123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146,132, 133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140,153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24,5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14,34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, or

(vi) is selected from the group of genes having a nucleotide sequenceencoding an amino acid sequence that, when the two amino acid sequencesare optimally aligned and compared, is at least 70% preferably at least75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the amino acid sequenceencoded by the nucleotide sequence represented in any of SEQ ID NOs 277,138, 253, 152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129,144, 130, 145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136,150, 276, 137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256,280, 1, 21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10,30, 11, 31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19,39, 20 or 40, or

(vii) is selected from the group of genes having a nucleotide sequenceencoding an amino acid sequence that, when the two amino acid sequencesare optimally aligned and compared, is at least 70% preferably at least75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the amino acid sequencerepresented in any of SEQ ID NOs 285, 242, 271, 226, 227, 281, 228, 282,229, 230 to 233, 234, 283, 235, 236, 237, 238, 239, 240, 284, 241, 243,244, 286, 269, 270, 287, 288, 206 to 225;

(b) regenerating a plant from the transformed plant cell; and

(c) growing the transformed plant under conditions suitable for theexpression of the interfering RNA from the recombinant DNA construct,said plant thus being resistant to said pest as compared with anuntransformed plant.

In accordance with a sixth aspect of the invention, there is provided atransgenic plant, or reproductive or propagation material for atransgenic plant or a cultured transgenic plant cell, which expresses oris capable of expressing at least one interfering ribonucleic acid (RNA)that functions upon uptake by an insect pest species to down-regulatethe expression of a target gene within said pest, wherein the targetgene

(i) is selected from the group of genes having a nucleotide sequencecomprising any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273,123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146, 132,133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140, 153,278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5,25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34,15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, or the complementthereof, or having a nucleotide sequence that, when the two sequencesare optimally aligned and compared, is at least 75%, preferably at least80%, 85%, 90%, 95%, 98% or 99% identical any of SEQ ID NOs 277, 138,253, 152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144,130, 145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150,276, 137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1,21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11,31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20or 40, or the complement thereof, or

(ii) is selected from the group of genes having a nucleotide sequenceconsisting of any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273,123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146, 132,133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140, 153,278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5,25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34,15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, or the complementthereof, or

(iii) is selected from the group of genes having a nucleotide sequencecomprising a fragment of at least 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 125, 150, 175,200, 225, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900 1000,1100 or 1115 contiguous nucleotides of any of SEQ ID NOs 277, 138, 253,152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144, 130,145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150, 276,137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21,2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31,12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or40, or the complement thereof, or having a nucleotide sequence that,when said gene comprising said fragment is optimally aligned andcompared with any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273,123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146, 132,133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140, 153,278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5,25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34,15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, said nucleotidesequence is at least 75% preferably at least 80%, 85%, 90%, 95%, 98% or99% identical to any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141,273, 123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146,132, 133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140,153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24,5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14,34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, or the complementthereof, or

(iv) is selected from the group of genes having a nucleotide sequencecomprising a fragment of at least 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 125, 150, 175,200, 225, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900 1000,1100 or 1115 contiguous nucleotides of any of SEQ ID NOs 277, 138, 253,152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144, 130,145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150, 276,137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21,2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31,12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or40, or the complement thereof, and wherein when said fragment isoptimally aligned and compared with the corresponding fragment in any ofSEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273, 123, 142, 274, 124,143, 125 to 129, 144, 130, 145, 275, 131, 146, 132, 133, 147, 134, 148,135, 149, 136, 150, 276, 137, 151, 139, 140, 153, 278, 251, 254, 279,252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8,28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17,37, 18, 38, 19, 39, 20 or 40, said nucleotide sequence of said fragmentis at least 75% preferably at least 80%, 85%, 90%, 95%, 98% or 99%identical to said corresponding fragment of any of SEQ ID NOs 277, 138,253, 152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144,130, 145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150,276, 137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1,21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11,31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20or 40, or the complement thereof, or

(v) is an insect pest orthologue of a gene having a nucleotide sequencecomprising any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273,123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146, 132,133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140, 153,278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5,25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34,15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, or the complementthereof, wherein the two orthologous genes are similar in sequence tosuch a degree that when the two genes are optimally aligned andcompared, the orthologue has a sequence that is at least 75% preferablyat least 80%, 85%, 90%, 95%, 98% or 99% identical to any of thesequences represented by SEQ ID NOs 277, 138, 253, 152, 121, 122, 141,273, 123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146,132, 133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140,153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24,5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14,34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, or

(vi) is selected from the group of genes having a nucleotide sequenceencoding an amino acid sequence that, when the two amino acid sequencesare optimally aligned and compared, is at least 70% preferably at least75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the amino acid sequenceencoded by the nucleotide sequence represented in any of SEQ ID NOs 277,138, 253, 152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129,144, 130, 145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136,150, 276, 137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256,280, 1, 21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10,30, 11, 31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19,39, 20 or 40, or

(vii) is selected from the group of genes having a nucleotide sequenceencoding an amino acid sequence that, when the two amino acid sequencesare optimally aligned and compared, is at least 70% preferably at least75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the amino acid sequencerepresented in any of SEQ ID NOs 285, 242, 271, 226, 227, 281, 228, 282,229, 230 to 233, 234, 283, 235, 236, 237, 238, 239, 240, 284, 241, 243,244, 286, 269, 270, 287, 288, 206 to 225.

In one embodiment, the present invention relates to a transgenic plantor plant cell comprising an interfering nucleic acid (RNA or doublestranded RNA) that is at least 75% preferably at least 80%, 85%, 90%,95%, 98%, 99% or 100% complementary to a part of at least 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70,80, 90, 100, 110, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450, 500,550, 600, 700, 800, 900 1000, 1100 or 1115 contiguous nucleotides of orall of a mRNA encoding an insect ribosomal protein such as the ribosomalprotein L19 (e.g. an insect orthologue of the CG2746 Dm protein), theribosomal protein L40 (e.g. an insect orthologue of the CG2960 Dmprotein) or the ribosomal protein S27A (e.g. an insect orthologue of theCG5271 Dm protein), and wherein the interfering nucleic acid inhibits orinterferes with the translation of said mRNA and wherein the plant orplant cell is resistant to the insect as compared with an untransformedplant.

According to another embodiment the present invention relates to atransgenic plant or plant cell comprising an interfering nucleic acid(RNA or double stranded RNA) that is at least 75% preferably at least80%, 85%, 90%, 95%, 98%, 99% or 100% complementary to a part of at least17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50,55, 60, 70, 80, 90, 100, 110, 125, 150, 175, 200, 225, 250, 300, 350,400, 450, 500, 550, 600, 700, 800, 900 1000, 1100 or 1115 contiguousnucleotides of or all of a mRNA encoding an insect proteasome subunitsuch as the Rpn6 protein (e.g. an insect orthologue of the CG10149 Dmprotein), the Pros 25 protein (e.g. an insect orthologue of the CG5266Dm protein), the Rpn2 protein (e.g. an insect orthologue of the CG11888Dm protein), the proteasome beta 1 subunit protein (e.g. an insectorthologue of the CG8392 Dm protein) or the Pros beta 2 protein (e.g. aninsect orthologue of the CG3329 Dm protein), and wherein the interferingnucleic acid inhibits or interferes with the translation of said mRNAand wherein the plant or plant cell is resistant to the insect ascompared with an untransformed plant.

According to still another embodiment the present invention relates to atransgenic plant or plant cell comprising an interfering nucleic acid(RNA or double stranded RNA) that is at least 75% preferably at least80%, 85%, 90%, 95%, 98%, 99% or 100% complementary to a part of at least17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50,55, 60, 70, 80, 90, 100, 110, 125, 150, 175, 200, 225, 250, 300, 350,400, 450, 500, 550, 600, 700, 800, 900 1000, 1100 or 1115 contiguousnucleotides of or all of a mRNA encoding an insect β-coatomer of theCOPI vesicle (e.g. an insect orthologue of the CG6223 Dm protein), theγ-coatomer of the COPI vesicle (e.g. an insect orthologue of the 1528 Dmprotein), the β′-coatomer protein (e.g. an insect orthologue of theCG6699 Dm protein) or the ζ-coatomer of the COPI vesicle (e.g. an insectorthologue of the CG3948 Dm protein), and wherein the interferingnucleic acid inhibits or interferes with the translation of said mRNAand wherein the plant or plant cell is resistant to the insect ascompared with an untransformed plant.

According to still another embodiment the present invention relates to atransgenic plant or plant cell comprising an interfering nucleic acid(RNA or double stranded RNA) that is at least 75% preferably at least80%, 85%, 90%, 95%, 98%, 99% or 100% complementary to a part of at least17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50,55, 60, 70, 80, 90, 100, 110, 125, 150, 175, 200, 225, 250, 300, 350,400, 450, 500, 550, 600, 700, 800, 900 1000, 1100 or 1115 contiguousnucleotides of or all of a mRNA encoding an insect Tetraspanine 2 Aprotein which is a putative transmembrane domain protein (e.g. an insectorthologue of the CG11415 Dm protein), and wherein the interferingnucleic acid inhibits or interferes with the translation of said mRNAand wherein the plant or plant cell is resistant to the insect ascompared with an untransformed plant.

According to still another embodiment the present invention relates to atransgenic plant or plant cell comprising an interfering nucleic acid(RNA or double stranded RNA) that is at least 75% preferably at least80%, 85%, 90%, 95%, 98%, 99% or 100% complementary to a part of at least17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50,55, 60, 70, 80, 90, 100, 110, 125, 150, 175, 200, 225, 250, 300, 350,400, 450, 500, 550, 600, 700, 800, 900 1000, 1100 or 1115 contiguousnucleotides of or all of a mRNA encoding an insect protein belonging tothe actin family (e.g. an insect orthologue of the CG5409 Dm protein)such as Actin 5C (e.g. an insect orthologue of the CG4027 Dm protein),and wherein the interfering nucleic acid inhibits or interferes with thetranslation of said mRNA and wherein the plant or plant cell isresistant to the insect as compared with an untransformed plant.

According to still another embodiment the present invention relates to atransgenic plant or plant cell comprising an interfering nucleic acid(RNA or double stranded RNA) that is at least 75% preferably at least80%, 85%, 90%, 95%, 98%, 99% or 100% complementary to a part of at least17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50,55, 60, 70, 80, 90, 100, 110, 125, 150, 175, 200, 225, 250, 300, 350,400, 450, 500, 550, 600, 700, 800, 900 1000, 1100 or 1115 contiguousnucleotides of or all of a mRNA encoding an insect ubiquitin-5E protein(e.g. an insect orthologue of the CG32744 Dm protein), and wherein theinterfering nucleic acid inhibits or interferes with the translation ofsaid mRNA and wherein the plant or plant cell is resistant to the insectas compared with an untransformed plant.

According to still another embodiment the present invention relates to atransgenic plant or plant cell comprising an interfering nucleic acid(RNA or double stranded RNA) that is at least 75% preferably at least80%, 85%, 90%, 95%, 98%, 99% or 100% complementary to a part of at least17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50,55, 60, 70, 80, 90, 100, 110, 125, 150, 175, 200, 225, 250, 300, 350,400, 450, 500, 550, 600, 700, 800, 900 1000, 1100 or 1115 contiguousnucleotides of or all of a mRNA encoding an insect Sec23 protein whichis a GTPase activator involved in intracellular protein transport (e.g.an insect orthologue of the CG1250 Dm protein), and wherein theinterfering nucleic acid inhibits or interferes with the translation ofsaid mRNA and wherein the plant or plant cell is resistant to the insectas compared with an untransformed plant.

According to still another embodiment the present invention relates to atransgenic plant or plant cell comprising an interfering nucleic acid(RNA or double stranded RNA) that is at least 75% preferably at least80%, 85%, 90%, 95%, 98%, 99% or 100% complementary to a part of at least17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50,55, 60, 70, 80, 90, 100, 110, 125, 150, 175, 200, 225, 250, 300, 350,400, 450, 500, 550, 600, 700, 800, 900 1000, 1100 or 1115 contiguousnucleotides of or all of a mRNA encoding an insect crinkled proteinwhich is an unconventional myosin which is involved in motor activity(e.g. an insect orthologue of the CG7595 Dm protein), and wherein theinterfering nucleic acid inhibits or interferes with the translation ofsaid mRNA and wherein the plant or plant cell is resistant to the insectas compared with an untransformed plant.

According to still another embodiment the present invention relates to atransgenic plant or plant cell comprising an interfering nucleic acid(RNA or double stranded RNA) that is at least 75% preferably at least80%, 85%, 90%, 95%, 98%, 99% or 100% complementary to a part of at least17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50,55, 60, 70, 80, 90, 100, 110, 125, 150, 175, 200, 225, 250, 300, 350,400, 450, 500, 550, 600, 700, 800, 900 1000, 1100 or 1115 contiguousnucleotides of or all of a mRNA encoding an insect crooked neck proteinwhich is involved in the regulation of nuclear alternative mRNA splicing(e.g. an insect orthologue of the CG3193 Dm protein), and wherein theinterfering nucleic acid inhibits or interferes with the translation ofsaid mRNA and wherein the plant or plant cell is resistant to the insectas compared with an untransformed plant.

According to still another embodiment the present invention relates to atransgenic plant or plant cell comprising an interfering nucleic acid(RNA or double stranded RNA) that is at least 75% preferably at least80%, 85%, 90%, 95%, 98%, 99% or 100% complementary to a part of at least17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50,55, 60, 70, 80, 90, 100, 110, 125, 150, 175, 200, 225, 250, 300, 350,400, 450, 500, 550, 600, 700, 800, 900 1000, 1100 or 1115 contiguousnucleotides of or all of a mRNA encoding an insect vacuolar H+-ATPaseG-subunit protein (e.g. an insect orthologue of the CG6213 Dm protein),and wherein the interfering nucleic acid inhibits or interferes with thetranslation of said mRNA and wherein the plant or plant cell isresistant to the insect as compared with an untransformed plant.

According to still another embodiment the present invention relates to atransgenic plant or plant cell comprising an interfering nucleic acid(RNA or double stranded RNA) that is at least 75% preferably at least80%, 85%, 90%, 95%, 98%, 99% or 100% complementary to a part of at least17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50,55, 60, 70, 80, 90, 100, 110, 125, 150, 175, 200, 225, 250, 300, 350,400, 450, 500, 550, 600, 700, 800, 900 1000, 1100 or 1115 contiguousnucleotides of or all of a mRNA encoding an insect Tbp-1; Tat-bindingprotein (e.g. an insect orthologue of the CG10370 Dm protein), andwherein the interfering nucleic acid inhibits or interferes with thetranslation of said mRNA and wherein the plant or plant cell isresistant to the insect as compared with an untransformed plant.

BRIEF DESCRIPTION OF THE TABLES AND FIGURES

Table 1 Polynucleotide sequences of target genes identified inLeptinotarsa decemlineata (Colorado potato beetle, CPB).

Table 2 Amino acid sequences of target genes identified in Leptinotarsadecemlineata (Colorado potato beetle, CPB).

Table 3 Polynucleotide sequences of target genes identified in Lygushesperus.

Table 4 Amino acid sequences of target genes identified in Lygushesperus.

Table 5 dsRNAs (sense strand represented by equivalent DNA sequence)corresponding to Leptinotarsa decemlineata target genes.

Table 6 dsRNAs (sense strand represented by equivalent DNA sequence)corresponding to Lygus hesperus target genes.

Table 7 Effects of dsRNAs derived from different target genes on timetaken to kill 50% (LT₅₀) of CPB larvae expressed as ratios versus theeffect of a dsRNA derived from reference target gene Ld248 (SEQ ID NO40).

Table 8 Effects of dsRNAs derived from different target genes on timetaken to kill 50% (LT₅₀) of CPB adults expressed as ratios versus theeffect of a dsRNA derived from reference target gene Ld248 (SEQ ID NO40).

Table 9 Effects of dsRNAs derived from different target genes on CPB eggproduction. Abbreviations: EM, normal egg masses; SE, single eggs; YS,yellow smear; none, no eggs.

Table 10 Survival analyses of target dsRNAs versus GFP dsRNA in thepresence of tRNA in a Lygus hesperus nymph feeding assay. Log-rank testused to test the differences between the survival curves of the targetdsRNA (or diet only) and GFP dsRNA generated using Kaplan-Meierestimates.

Table 11 Ranking of different target genes according to potency.

Table 12 Comparison of survival curves of test targets at 0.1, 0.05, or0.025 μg/μL with GFP dsRNA at 0.1 μg/μL. Statistics were performed ondata graphically represented in FIG. 5. ***: P-value ≤0.001; **:0.001<P-value ≤0.01; *: 0.01<P-value ≤0.05; ns: not significant, P-value>0.05.

Table 13 Lygus targets for which full length cDNAs were cloned.

Table 14 Full length polynucleotide sequences of target genes identifiedin Lygus hesperus.

Table 15 Corresponding amino acid sequences to full length cDNAs oftarget genes identified in Lygus hesperus.

FIG. 1 Effects of dsRNAs derived from different target genes on survivaland fitness of CPB adults. For each target gene investigated, 10 youngadult beetles were individually fed target dsRNA-treated potato leafdiscs (total of 10 μg dsRNA) for the first 24 hours and thereafterplaced together on untreated potato foliage. The numbers of dead ormoribund insects were assessed over a 14-day period. Data are presentedas percentage mortality or moribundity. GFP dsRNA (SEQ ID NO 245) wasused as a control. Ld105 dsRNA (SEQ ID NO 39), Ld248 dsRNA (SEQ ID NO40), Ld516 dsRNA (SEQ ID NO 29), Ld511 dsRNA (SEQ ID NO 36), Ld512 dsRNA(SEQ ID NO 37), Ld513 dsRNA (SEQ ID NO 22), Ld520 dsRNA (SEQ ID NO 24),Ld537 dsRNA (SEQ ID NO 25), Ld563 dsRNA (SEQ ID NO 38), Ld579 dsRNA (SEQID NO 30).

FIG. 2 Survival curves for Lygus hesperus nymphs exposed to 0.5 μg/μLtarget dsRNA in the presence of 5 μg/μL yeast tRNA in a feeding assay.(a) Targets: Lh520 (SEQ ID NO 143), Lh423 (SEQ ID NO 152), Lh537 (SEQ IDNO 144), (b) Targets: Lh504.2 (SEQ ID NO 142), Lh512 (SEQ ID NO 153),Lh334 (SEQ ID NO 145), (c) Targets: Lh300.1 (SEQ ID NO 151), Lh327 (SEQID NO 146), Lh332 (SEQ ID NO 148), (d) Targets: Lh237 (SEQ ID NO 149),Lh579 (SEQ ID NO 147), Lh261 (SEQ ID NO 150), Lh513 (SEQ ID NO 141). GFPdsRNA plus yeast tRNA at the same concentrations, respectively, anddiet-only treatments were used as controls. Young nymphs were eachexposed to 25 μL of 15% sucrose diet with or without incorporated testcomponents for three days prior to transferring them on to 50 μL Bioservdiet. Complex diet was refreshed on day 7. For all treatments, n=24.

FIG. 3 Survival curves for Lygus hesperus nymphs exposed to 0.5 μg/μLtarget dsRNA in the presence of 5 μg/μL yeast tRNA in a feeding assay,wherein the targets are grouped in A, B, C, D according to potency.Set-up described as in FIG. 2.

FIG. 4 Survival curves over time of Lygus hesperus nymphs exposed tolowering concentrations (from 0.5 to 0.1 μg/μL) of novel target dsRNA inthe presence of yeast transfer RNA (5 μg/μL) in feeding bioassays. Eachtreatment in a bioassay consisted of 24 one-day-old nymphs placedindividually in every well of a 24-well plate. Each nymph was exposed toa parafilm sachet containing the ribonucleic acids in a solution of 15%sucrose for a duration of 3 days. On days 3 and 7, the diets werereplaced with fresh rearing (Bioserv) diet. The following controls wereincluded in the assays: GFP dsRNA and diet only.

FIG. 5 Survival curves over time of Lygus hesperus nymphs exposed tolowering concentrations (from 0.1 to 0.025 μg/μL) of novel target dsRNAin the presence of yeast transfer RNA (5 μg/μL) in feeding bioassays.Set-up described similarly as in FIG. 4.

FIG. 6 Survival curves over time of Lygus hesperus nymphs exposed to 0.5μg/μL of target dsRNA in the presence of yeast transfer RNA (5 μg/μL) infeeding bioassays. Target dsRNA tested: Lh105.2 (SEQ ID NO 254), Lh248.2(SEQ ID NO 255), Lh248.3 (SEQ ID NO 256), Lh327 (SEQ ID NO 146) andLh300 (SEQ ID NO 151). Set-up described similarly as in FIG. 4.

FIG. 7 Schematic representation of the plant expression vectorharbouring the Lygus hesperus hpRNA cassette. RB: right border; LB: leftborder; P35S: Cauliflower Mosaic Virus 35S promoter; T35S: CauliflowerMosaic Virus 35S terminator; TNOS: nopaline synthase terminator; GFP:green fluorescent reporter gene; NPT II: coding sequence of neomycinphosphotransferase II gene; KmR: Kanamycin resistance gene; pBR322 ori:pBR322 origin of replication; pBR322 bom: pBR322 mobilization; pVS1 rep:pVS1 replicon; pVS1 sta: pVS1 stability element.

FIG. 8: Potato-Lygus in planta assay set up. White arrows indicateinsect damage.

FIG. 9 Lygus feeding assays on transgenic potatoes, expressing Lh423hairpin. Survival rate of Lygus nymphs feeding on transgenic potatoescarrying Lh423 hairpin (P006/XX) or a GUS hairpin (P001/XX). Wild type(WT) potatoes were also used as control.

FIG. 10 Lygus feeding assays on positive transgenic potatoes, expressingLh423 hairpin. Survival rate of Lygus nymphs feeding on transgenicpotatoes carrying Lh423 hairpin (P006/59, P006/29 and P006/22) or a GUShairpin (P001/19, P001/28). Wild type (WT) potatoes were also used ascontrol. Statistical analysis results, based on GraphPad survival curveanalysis: ***=P<0.001; *=0.01<P<0.05.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have discovered that down-regulating theexpression of particular target genes in insect pest species by RNAi canbe used to effectively prevent and/or control infestation by said insectpest. As used herein, the term “control” of pest infestation refers toany effect on a pest that serves to limit and/or reduce either thenumbers of pest organisms and/or the damage caused by the pest.Preferred target genes are therefore essential genes that control orregulate one or more essential biological functions within the insectpest, for example, cell division, reproduction, energy metabolism,digestion, neurological function and the like. Down-regulation of theseessential genes by RNAi techniques can lead to death of the insect, orotherwise significantly retard growth and development or impair theability of the pest to colonize an environment or infest host organisms.

Thus, in a first aspect, the invention provides an interferingribonucleic acid (RNA) that functions upon uptake by an insect pestspecies to down-regulate expression of a target gene in said insectpest.

As used herein, a “target gene” comprises any gene in the insect pestwhich one intends to down-regulate. In a preferred embodiment, thetarget gene is down-regulated so as to control pest infestation, forexample by disrupting an essential biological process occurring in thepest, or by decreasing the pathogenicity of the pest. Preferred targetgenes therefore include but are not limited to those that play key rolesin regulating feeding, survival, growth, development, reproduction,infestation and infectivity. According to one embodiment, the targetgene is such that when its expression is down-regulated or inhibited,the insect pest is killed. According to another embodiment, the targetgene is such that when its expression is down-regulated or inhibited,growth of the pest is prevented or retarded or stunted or delayed orimpeded or pest reproduction is prevented. According to yet anotherembodiment of the invention, the target gene is such that when itsexpression is down-regulated or inhibited, the damage caused by the pestand/or the ability of the pest to infect or infest environments,surfaces and/or plant or crop species is reduced; or the pest stopsfeeding from its natural food resources such as plants and plantproducts. The terms “infest” and “infect” or “infestation” and“infection” are generally used interchangeably throughout.

The target genes may be expressed in all or some of the cells of theinsect pest. Furthermore, the target genes may only be expressed by theinsect pest at a particular stage of its life-cycle, for example, themature adult phase, immature nymph or larval phase or egg phase.

In specific embodiments, the present invention provides target geneswhich encode proteins involved in the function of a proteasome (subunitor regulatory particle), ribosomal protein, intracellular proteintransport, COPI vesicle (coat protein complex), protein modificationprocess, cytoskeleton, ATPase or GTPase activator activity (specified inTables 7 and 8).

In preferred embodiments, the present invention provides target genesselected from the group of genes having a nucleotide sequence comprisingany of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273, 123, 142, 274,124, 143, 125 to 129, 144, 130, 145, 275, 131, 146, 132, 133, 147, 134,148, 135, 149, 136, 150, 276, 137, 151, 139, 140, 153, 278, 251, 254,279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7,27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34, 15, 35, 16,36, 17, 37, 18, 38, 19, 39, 20 or 40, or the complement thereof, orhaving a nucleotide sequence that, when the two sequences are optimallyaligned and compared, is at least 75%, preferably at least 80%, 85%,90%, 95%, 98% or 99% identical any of SEQ ID NOs 277, 138, 253, 152,121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144, 130, 145,275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150, 276, 137,151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2,22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12,32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, orthe complement thereof, or having a nucleotide sequence consisting ofany of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273, 123, 142, 274,124, 143, 125 to 129, 144, 130, 145, 275, 131, 146, 132, 133, 147, 134,148, 135, 149, 136, 150, 276, 137, 151, 139, 140, 153, 278, 251, 254,279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7,27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34, 15, 35, 16,36, 17, 37, 18, 38, 19, 39, 20 or 40, or the complement thereof, orhaving a nucleotide sequence comprising a fragment of at least 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90,100, 110, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450, 500, 550,600, 700, 800, 900 1000, 1100 or 1115 contiguous nucleotides of any ofSEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273, 123, 142, 274, 124,143, 125 to 129, 144, 130, 145, 275, 131, 146, 132, 133, 147, 134, 148,135, 149, 136, 150, 276, 137, 151, 139, 140, 153, 278, 251, 254, 279,252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8,28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17,37, 18, 38, 19, 39, 20 or 40, or the complement thereof, or having anucleotide sequence that, when said gene comprising said fragment isoptimally aligned and compared with any of SEQ ID NOs 277, 138, 253,152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144, 130,145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150, 276,137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21,2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31,12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or40, said nucleotide sequence is at least 75% preferably at least 80%,85%, 90%, 95%, 98% or 99% identical to any of SEQ ID NOs 277, 138, 253,152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144, 130,145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150, 276,137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21,2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31,12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or40, or the complement thereof, or having a nucleotide sequencecomprising a fragment of at least 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 125, 150, 175,200, 225, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900 1000,1100 or 1115 contiguous nucleotides of any of SEQ ID NOs 277, 138, 253,152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144, 130,145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150, 276,137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21,2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31,12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or40, or the complement thereof, and wherein when said fragment isoptimally aligned and compared with the corresponding fragment in any ofSEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273, 123, 142, 274, 124,143, 125 to 129, 144, 130, 145, 275, 131, 146, 132, 133, 147, 134, 148,135, 149, 136, 150, 276, 137, 151, 139, 140, 153, 278, 251, 254, 279,252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8,28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17,37, 18, 38, 19, 39, 20 or 40, said nucleotide sequence of said fragmentis at least 75% preferably at least 80%, 85%, 90%, 95%, 98% or 99%identical to said corresponding fragment of any of SEQ ID NOs 277, 138,253, 152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144,130, 145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150,276, 137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1,21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11,31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20or 40, or the complement thereof, or which gene is an insect pestorthologue of a gene having a nucleotide sequence comprising any of SEQID NOs 277, 138, 253, 152, 121, 122, 141, 273, 123, 142, 274, 124, 143,125 to 129, 144, 130, 145, 275, 131, 146, 132, 133, 147, 134, 148, 135,149, 136, 150, 276, 137, 151, 139, 140, 153, 278, 251, 254, 279, 252,255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28,9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37,18, 38, 19, 39, 20 or 40, or the complement thereof, wherein the twoorthologous genes are similar in sequence to such a degree that when thetwo genes are optimally aligned and compared, the orthologue has asequence that is at least 75% preferably at least 80%, 85%, 90%, 95%,98% or 99% identical to any of the sequences represented by SEQ ID NOs277, 138, 253, 152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to129, 144, 130, 145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149,136, 150, 276, 137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255,256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29,10, 30, 11, 31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38,19, 39, 20 or 40, or having a nucleotide sequence encoding an amino acidsequence that, when the two amino acid sequences are optimally alignedand compared, is at least 70% preferably at least 75%, 80%, 85%, 90%,95%, 98% or 99% identical to the amino acid sequence encoded by thenucleotide sequence represented by any of SEQ ID NOs 277, 138, 253, 152,121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144, 130, 145,275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150, 276, 137,151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2,22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12,32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40 orhaving a nucleotide sequence encoding an amino acid sequence that, whenthe two amino acid sequences are optimally aligned and compared, is atleast 70% preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99%identical to the amino acid sequence represented in any of SEQ ID NOs285, 242, 271, 226, 227, 281, 228, 282, 229, 230 to 233, 234, 283, 235,236, 237, 238, 239, 240, 284, 241, 243, 244, 286, 269, 270, 287, 288,206 to 225; and wherein the nucleotide sequence of said gene is nolonger than 5000, 4000, 3000, 2000 or 1500 nucleotides.

As used herein, the term “sequence identity” is used to describe thesequence relationship between two or more nucleotide or amino acidsequences. The percentage of “sequence identity” between two sequencesis determined by comparing two optimally aligned sequences over acomparison window (a defined number of positions), wherein the portionof the sequence in the comparison window may comprise additions ordeletions (i.e. gaps) as compared to the reference sequence in order toachieve optimal alignment. The percentage sequence identity iscalculated by determining the number of positions at which the identicalnucleotide base or amino acid residue occurs in both sequences to yieldthe number of ‘matched’ positions, dividing the number of matchedpositions by the total number of positions in the comparison window andmultiplying the result by 100. For comparison of two optimally alignedsequences, the comparison window will be determined by the full lengthof the aligned regions. Methods and software for determining sequenceidentity are available in the art and include the Blast software and GAPanalysis. For nucleic acids, the percent identity is calculatedpreferably by the BlastN alignment tool whereby the percent identity iscalculated over the entire length of the query nucleotide sequence.

A person skilled in the art will recognise that homologues ororthologues (homologues existing in different species) of the targetgenes represented by any of SEQ ID NOs 277, 138, 253, 152, 121, 122,141, 273, 123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131,146, 132, 133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139,140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23,4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13,33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40 can beidentified. These pest homologues and/or orthologues are also within thescope of the current invention. Preferred homologues and/or orthologuesare genes similar in sequence to such a degree that when the two genesare optimally aligned and compared, the homologue and/or orthologue hasa sequence that is at least 75%, preferably at least 80% or 85%, morepreferably at least 90% or 95%, and most preferably at least about 99%identical to any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273,123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146, 132,133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140, 153,278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5,25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34,15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40 or the complementthereof.

Other homologues are genes which are alleles of a gene comprising asequence as represented by any of SEQ ID NOs 277, 138, 253, 152, 121,122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275,131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151,139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3,23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32,13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40.Further preferred homologues are genes comprising at least one singlenucleotide polymorphism (SNP) compared to a gene comprising a sequenceas represented by any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141,273, 123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146,132, 133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140,153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24,5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14,34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40.

The ‘interfering ribonucleic acid (RNA)’ of the current inventionencompasses any type of RNA molecule capable of down-regulating or‘silencing’ expression of a target gene, including but not limited tosense RNA, antisense RNA, short interfering RNA (siRNA), microRNA(miRNA), double-stranded RNA (dsRNA), short hairpin RNA (shRNA) and thelike. Methods to assay for functional interfering RNA molecules are wellknown in the art and are disclosed elsewhere herein.

The interfering RNA molecules of the current invention effectsequence-specific down-regulation of expression of a target gene bybinding to a target nucleotide sequence within the target gene. Bindingoccurs as a result of base pairing between complementary regions of theinterfering RNA and the target nucleotide sequence. As used herein, theterm ‘silencing element’ refers to the portion or region of theinterfering RNA comprising or consisting of a sequence of nucleotideswhich is complementary, or at least partially complementary, to a targetnucleotide sequence within the target gene, and which functions as theactive portion of the interfering RNA to direct down-regulation ofexpression of said target gene. In one embodiment of the invention, thesilencing element comprises or consists of a sequence of at least 17contiguous nucleotides, preferably at least 18 or 19 contiguousnucleotides, more preferably at least 21 contiguous nucleotides, evenmore preferably at least 22, 23, 24 or 25 contiguous nucleotidescomplementary to a target nucleotide sequence within the target gene.

As used herein, “expression of a target gene” refers to thetranscription and accumulation of the RNA transcript encoded by a targetgene and/or translation of the mRNA into protein. The term‘down-regulate’ is intended to refer to any of the methods known in theart by which interfering RNA molecules reduce the level of primary RNAtranscripts, mRNA or protein produced from a target gene. In certainembodiments, down-regulation refers to a situation whereby the level ofRNA or protein produced from a gene is reduced by at least 10%,preferably by at least 33%, more preferably by at least 50%, yet morepreferably by at least 80%. In particularly preferred embodiments,down-regulation refers to a reduction in the level of RNA or proteinproduced from a gene by at least 80%, preferably by at least 90%, morepreferably by at least 95%, and most preferably by at least 99% withincells of the insect pest as compared with an appropriate control insectpest which has for example, not been exposed to an interfering RNA orhas been exposed to a control interfering RNA molecule. Methods fordetecting reductions in RNA or protein levels are well known in the artand include RNA solution hybridization, Northern hybridization, reversetranscription (e.g. quantitative RT-PCR analysis), microarray analysis,antibody binding, enzyme-linked immunosorbent assay (ELISA) and Westernblotting. In another embodiment of the invention, down-regulation refersto a reduction in RNA or protein levels sufficient to result in adetectable change in a phenotype of the pest as compared with anappropriate pest control, for example, cell death, cessation of growth,or the like. Down-regulation can thus be measured by phenotypic analysisof the insect pest using techniques routine in the art.

In a preferred embodiment of the invention, the interfering RNAdown-regulates gene expression by RNA interference or RNAi. RNAi is aprocess of sequence-specific gene regulation typically mediated bydouble-stranded RNA molecules such as short interfering RNAs (siRNAs).siRNAs comprise a sense RNA strand annealed by complementary basepairingto an antisense RNA strand. The sense strand or ‘guide strand’ of thesiRNA molecule comprises a sequence of nucleotides complementary to asequence of nucleotides located within the RNA transcript of the targetgene. The sense strand of the siRNA is therefore able to anneal to theRNA transcript via Watson-Crick-type basepairing and target the RNA fordegradation within a cellular complex known as the RNAi-inducedsilencing complex or RISC. Thus, in the context of preferred interferingRNA molecules of the current invention, the silencing element asreferred to herein may be a double-stranded region comprising annealedcomplementary strands, at least one strand of which comprises orconsists of a sequence of nucleotides which is complementary or at leastpartially complementary to a target nucleotide sequence within a targetgene. In one embodiment the double-stranded region has a length of atleast 21, 22, 23, 24, 25, 30, 35, 40, 50, 55, 60, 70, 80, 90, 100, 125,150, 175, 200 base pairs.

Longer double-stranded RNA (dsRNA) molecules comprising one or morefunctional double-stranded silencing elements as described elsewhereherein, and capable of RNAi-mediated gene silencing are alsocontemplated within the scope of the current invention. Such longerdsRNA molecules comprise at least 80, 200, 300, 350, 400, 450, 500, 550,600, 700, 800, 900, 1000 or 1100 base pairs. These dsRNA molecules mayserve as precursors for the active siRNA molecules that direct the RNAtranscript to the RISC complex for subsequent degradation. dsRNAmolecules present in the environment surrounding an organism or thecells thereof may be taken up by the organism and processed by an enzymecalled Dicer to yield siRNA molecules. Alternatively, the dsRNA may beproduced in vivo i.e. transcribed from a polynucleotide orpolynucleotides encoding the same present within a cell, for instance abacterial cell or a plant cell, and subsequently processed by Dicereither within the host cell or preferably within the insect pest cellsfollowing uptake of the longer precursor dsRNA. The dsRNA may be formedfrom two separate (sense and antisense) RNA strands that anneal byvirtue of complementary basepairing. Alternatively, the dsRNA may be asingle strand that is capable of folding back on itself to form a shorthairpin RNA (shRNA) or stem-loop structure. In the case of a shRNA, thedouble-stranded region or ‘stem’ is formed from two regions or segmentsof the RNA that are essentially inverted repeats of one another andpossess sufficient complementarity to allow the formation of adouble-stranded region. One or more functional double-stranded silencingelements may be present in this ‘stem region’ of the molecule. Theinverted repeat regions are typically separated by a region or segmentof the RNA known as the ‘loop’ region. This region can comprise anynucleotide sequence conferring enough flexibility to allow self-pairingto occur between the flanking complementary regions of the RNA. Ingeneral, the loop region is substantially single-stranded and acts as aspacer element between the inverted repeats.

All the interfering RNA molecules of the invention effectsequence-specific down-regulation of expression of a target gene bybinding to a target nucleotide sequence within the target gene. Bindingoccurs as a result of complementary base pairing between the silencingelement of the interfering RNA and the target nucleotide sequence. Inone embodiment of the current invention, the target nucleotide sequencecomprises a sequence of nucleotides as represented by the RNA transcriptof the target gene, or a fragment thereof wherein the fragment ispreferably at least 17 nucleotides, more preferably at least 18, 19 or20 nucleotides, or most preferably at least 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 125, 150, 175,200, 225, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, 1000 or1100 nucleotides. In a preferred embodiment of the current invention,the target nucleotide sequence comprises a sequence of nucleotidesequivalent to the RNA transcript encoded by any of the polynucleotidesselected from the group consisting of (i) a polynucleotide whichcomprises at least 21, preferably at least 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 125, 150, 175,200, 225, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, 1000,1100 or 1115 contiguous nucleotides of a nucleotide sequence asrepresented by any of SEQ ID NOs 277, 138, 253, 152, 198 to 201, 121,122, 141, 154 to 157, 273, 123, 142, 158 to 161, 274, 124, 143, 162 to165, 125 to 129, 144, 166 to 169, 130, 145, 170 to 173, 275, 131, 146,174 to 177, 132, 133, 147, 178 to 181, 134, 148, 182 to 185, 135, 149,186 to 189, 136, 150, 190 to 193, 276, 137, 151, 194 to 197, 139, 140,153, 202 to 205, 278, 251, 254, 257 to 260, 279, 252, 255, 256, 261 to268, 280, 1, 21, 41 to 44, 2, 22, 45 to 48, 3, 23, 49 to 52, 4, 24, 53to 56, 5, 25, 57 to 60, 6, 26, 61 to 64, 7, 27, 65 to 68, 8, 28, 69 to72, 9, 29, 73 to 76, 10, 30, 77 to 80, 11, 31, 81 to 84, 12, 32, 85 to88, 13, 33, 89 to 92, 14, 34, 93 to 96, 15, 35, 97 to 100, 16, 36, 101to 104, 17, 37, 105 to 108, 18, 38, 109 to 112, 19, 39, 113 to 116, 20,40, 117 to 120, or the complement thereof, or (ii) a polynucleotidewhich consists of at least 21, preferably at least 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 125, 150,175, 200, 225, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900,1000, 1100 or 1115 contiguous nucleotides of a nucleotide sequence asrepresented by any of SEQ ID NOs 277, 138, 253, 152, 198 to 201, 121,122, 141, 154 to 157, 273, 123, 142, 158 to 161, 274, 124, 143, 162 to165, 125 to 129, 144, 166 to 169, 130, 145, 170 to 173, 275, 131, 146,174 to 177, 132, 133, 147, 178 to 181, 134, 148, 182 to 185, 135, 149,186 to 189, 136, 150, 190 to 193, 276, 137, 151, 194 to 197, 139, 140,153, 202 to 205, 278, 251, 254, 257 to 260, 279, 252, 255, 256, 261 to268, 280, 1, 21, 41 to 44, 2, 22, 45 to 48, 3, 23, 49 to 52, 4, 24, 53to 56, 5, 25, 57 to 60, 6, 26, 61 to 64, 7, 27, 65 to 68, 8, 28, 69 to72, 9, 29, 73 to 76, 10, 30, 77 to 80, 11, 31, 81 to 84, 12, 32, 85 to88, 13, 33, 89 to 92, 14, 34, 93 to 96, 15, 35, 97 to 100, 16, 36, 101to 104, 17, 37, 105 to 108, 18, 38, 109 to 112, 19, 39, 113 to 116, 20,40, 117 to 120, or the complement thereof,

Or (iii) a polynucleotide which comprises at least 21, preferably atleast 22, 23 or 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70,80, 90, 100, 110, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450, 500,550, 600, 700, 800, 900, 1000, 1100 or 1115 contiguous nucleotides of anucleotide sequence as represented in any of SEQ ID NOs 277, 138, 253,152, 198 to 201, 121, 122, 141, 154 to 157, 273, 123, 142, 158 to 161,274, 124, 143, 162 to 165, 125 to 129, 144, 166 to 169, 130, 145, 170 to173, 275, 131, 146, 174 to 177, 132, 133, 147, 178 to 181, 134, 148, 182to 185, 135, 149, 186 to 189, 136, 150, 190 to 193, 276, 137, 151, 194to 197, 139, 140, 153, 202 to 205, 278, 251, 254, 257 to 260, 279, 252,255, 256, 261 to 268, 280, 1, 21, 41 to 44, 2, 22, 45 to 48, 3, 23, 49to 52, 4, 24, 53 to 56, 5, 25, 57 to 60, 6, 26, 61 to 64, 7, 27, 65 to68, 8, 28, 69 to 72, 9, 29, 73 to 76, 10, 30, 77 to 80, 11, 31, 81 to84, 12, 32, 85 to 88, 13, 33, 89 to 92, 14, 34, 93 to 96, 15, 35, 97 to100, 16, 36, 101 to 104, 17, 37, 105 to 108, 18, 38, 109 to 112, 19, 39,113 to 116, 20, 40, 117 to 120, or the complement thereof, that, whenthe two sequences are optimally aligned and compared, saidpolynucleotide is at least 75% preferably at least 80%, 85%, 90%, 95%,98% or 99% identical to any of SEQ ID NOs 277, 138, 253, 152, 198 to201, 121, 122, 141, 154 to 157, 273, 123, 142, 158 to 161, 274, 124,143, 162 to 165, 125 to 129, 144, 166 to 169, 130, 145, 170 to 173, 275,131, 146, 174 to 177, 132, 133, 147, 178 to 181, 134, 148, 182 to 185,135, 149, 186 to 189, 136, 150, 190 to 193, 276, 137, 151, 194 to 197,139, 140, 153, 202 to 205, 278, 251, 254, 257 to 260, 279, 252, 255,256, 261 to 268, 280, 1, 21, 41 to 44, 2, 22, 45 to 48, 3, 23, 49 to 52,4, 24, 53 to 56, 5, 25, 57 to 60, 6, 26, 61 to 64, 7, 27, 65 to 68, 8,28, 69 to 72, 9, 29, 73 to 76, 10, 30, 77 to 80, 11, 31, 81 to 84, 12,32, 85 to 88, 13, 33, 89 to 92, 14, 34, 93 to 96, 15, 35, 97 to 100, 16,36, 101 to 104, 17, 37, 105 to 108, 18, 38, 109 to 112, 19, 39, 113 to116, 20, 40, 117 to 120, or the complement thereof, or (iv) apolynucleotide which comprises a fragment of at least 21, preferably atleast 22, 23 or 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70,80, 90, 100, 110, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450, 500,550, 600, 700, 800, 900, 1000, 1100 or 1115 contiguous nucleotides of anucleotide as represented in any of SEQ ID NOs 277, 138, 253, 152, 198to 201, 121, 122, 141, 154 to 157, 273, 123, 142, 158 to 161, 274, 124,143, 162 to 165, 125 to 129, 144, 166 to 169, 130, 145, 170 to 173, 275,131, 146, 174 to 177, 132, 133, 147, 178 to 181, 134, 148, 182 to 185,135, 149, 186 to 189, 136, 150, 190 to 193, 276, 137, 151, 194 to 197,139, 140, 153, 202 to 205, 278, 251, 254, 257 to 260, 279, 252, 255,256, 261 to 268, 280, 1, 21, 41 to 44, 2, 22, 45 to 48, 3, 23, 49 to 52,4, 24, 53 to 56, 5, 25, 57 to 60, 6, 26, 61 to 64, 7, 27, 65 to 68, 8,28, 69 to 72, 9, 29, 73 to 76, 10, 30, 77 to 80, 11, 31, 81 to 84, 12,32, 85 to 88, 13, 33, 89 to 92, 14, 34, 93 to 96, 15, 35, 97 to 100, 16,36, 101 to 104, 17, 37, 105 to 108, 18, 38, 109 to 112, 19, 39, 113 to116, 20, 40, 117 to 120, or the complement thereof, and wherein saidfragment or said complement has a nucleotide sequence that, when saidfragment is optimally aligned and compared with the correspondingfragment in any of SEQ ID NOs 277, 138, 253, 152, 198 to 201, 121, 122,141, 154 to 157, 273, 123, 142, 158 to 161, 274, 124, 143, 162 to 165,125 to 129, 144, 166 to 169, 130, 145, 170 to 173, 275, 131, 146, 174 to177, 132, 133, 147, 178 to 181, 134, 148, 182 to 185, 135, 149, 186 to189, 136, 150, 190 to 193, 276, 137, 151, 194 to 197, 139, 140, 153, 202to 205, 278, 251, 254, 257 to 260, 279, 252, 255, 256, 261 to 268, 280,1, 21, 41 to 44, 2, 22, 45 to 48, 3, 23, 49 to 52, 4, 24, 53 to 56, 5,25, 57 to 60, 6, 26, 61 to 64, 7, 27, 65 to 68, 8, 28, 69 to 72, 9, 29,73 to 76, 10, 30, 77 to 80, 11, 31, 81 to 84, 12, 32, 85 to 88, 13, 33,89 to 92, 14, 34, 93 to 96, 15, 35, 97 to 100, 16, 36, 101 to 104, 17,37, 105 to 108, 18, 38, 109 to 112, 19, 39, 113 to 116, 20, 40, 117 to120, said nucleotide sequence is at least 75% preferably at least 80%,85%, 90%, 95%, 98% or 99% identical to said corresponding fragment ofany of SEQ ID NOs 277, 138, 253, 152, 198 to 201, 121, 122, 141, 154 to157, 273, 123, 142, 158 to 161, 274, 124, 143, 162 to 165, 125 to 129,144, 166 to 169, 130, 145, 170 to 173, 275, 131, 146, 174 to 177, 132,133, 147, 178 to 181, 134, 148, 182 to 185, 135, 149, 186 to 189, 136,150, 190 to 193, 276, 137, 151, 194 to 197, 139, 140, 153, 202 to 205,278, 251, 254, 257 to 260, 279, 252, 255, 256, 261 to 268, 280, 1, 21,41 to 44, 2, 22, 45 to 48, 3, 23, 49 to 52, 4, 24, 53 to 56, 5, 25, 57to 60, 6, 26, 61 to 64, 7, 27, 65 to 68, 8, 28, 69 to 72, 9, 29, 73 to76, 10, 30, 77 to 80, 11, 31, 81 to 84, 12, 32, 85 to 88, 13, 33, 89 to92, 14, 34, 93 to 96, 15, 35, 97 to 100, 16, 36, 101 to 104, 17, 37, 105to 108, 18, 38, 109 to 112, 19, 39, 113 to 116, 20, 40, 117 to 120 orthe complement thereof,

or (v) a polynucleotide which consists of a fragment of at least 21,preferably at least 22, 23 or 24, 25, 26, 27, 28, 29, 30, 35, 40, 45,50, 55, 60, 70, 80, 90, 100, 110, 125, 150, 175, 200, 225, 250, 300,350, 400, 450, 500, 550, 600, 700, 800, 900, 1000, 1100 or 1115contiguous nucleotides of a nucleotide as represented in any of SEQ IDNOs 277, 138, 253, 152, 198 to 201, 121, 122, 141, 154 to 157, 273, 123,142, 158 to 161, 274, 124, 143, 162 to 165, 125 to 129, 144, 166 to 169,130, 145, 170 to 173, 275, 131, 146, 174 to 177, 132, 133, 147, 178 to181, 134, 148, 182 to 185, 135, 149, 186 to 189, 136, 150, 190 to 193,276, 137, 151, 194 to 197, 139, 140, 153, 202 to 205, 278, 251, 254, 257to 260, 279, 252, 255, 256, 261 to 268, 280, 1, 21, 41 to 44, 2, 22, 45to 48, 3, 23, 49 to 52, 4, 24, 53 to 56, 5, 25, 57 to 60, 6, 26, 61 to64, 7, 27, 65 to 68, 8, 28, 69 to 72, 9, 29, 73 to 76, 10, 30, 77 to 80,11, 31, 81 to 84, 12, 32, 85 to 88, 13, 33, 89 to 92, 14, 34, 93 to 96,15, 35, 97 to 100, 16, 36, 101 to 104, 17, 37, 105 to 108, 18, 38, 109to 112, 19, 39, 113 to 116, 20, 40, 117 to 120, or the complementthereof, and wherein said fragment or said complement has a nucleotidesequence that, when said fragment is optimally aligned and compared withthe corresponding fragment in any of SEQ ID NOs 277, 138, 253, 152, 198to 201, 121, 122, 141, 154 to 157, 273, 123, 142, 158 to 161, 274, 124,143, 162 to 165, 125 to 129, 144, 166 to 169, 130, 145, 170 to 173, 275,131, 146, 174 to 177, 132, 133, 147, 178 to 181, 134, 148, 182 to 185,135, 149, 186 to 189, 136, 150, 190 to 193, 276, 137, 151, 194 to 197,139, 140, 153, 202 to 205, 278, 251, 254, 257 to 260, 279, 252, 255,256, 261 to 268, 280, 1, 21, 41 to 44, 2, 22, 45 to 48, 3, 23, 49 to 52,4, 24, 53 to 56, 5, 25, 57 to 60, 6, 26, 61 to 64, 7, 27, 65 to 68, 8,28, 69 to 72, 9, 29, 73 to 76, 10, 30, 77 to 80, 11, 31, 81 to 84, 12,32, 85 to 88, 13, 33, 89 to 92, 14, 34, 93 to 96, 15, 35, 97 to 100, 16,36, 101 to 104, 17, 37, 105 to 108, 18, 38, 109 to 112, 19, 39, 113 to116, 20, 40, 117 to 120, said nucleotide sequence is at least 75%preferably at least 80%, 85%, 90%, 95%, 98% or 99% identical to saidcorresponding fragment of any of SEQ ID NOs 277, 138, 253, 152, 198 to201, 121, 122, 141, 154 to 157, 273, 123, 142, 158 to 161, 274, 124,143, 162 to 165, 125 to 129, 144, 166 to 169, 130, 145, 170 to 173, 275,131, 146, 174 to 177, 132, 133, 147, 178 to 181, 134, 148, 182 to 185,135, 149, 186 to 189, 136, 150, 190 to 193, 276, 137, 151, 194 to 197,139, 140, 153, 202 to 205, 278, 251, 254, 257 to 260, 279, 252, 255,256, 261 to 268, 280, 1, 21, 41 to 44, 2, 22, 45 to 48, 3, 23, 49 to 52,4, 24, 53 to 56, 5, 25, 57 to 60, 6, 26, 61 to 64, 7, 27, 65 to 68, 8,28, 69 to 72, 9, 29, 73 to 76, 10, 30, 77 to 80, 11, 31, 81 to 84, 12,32, 85 to 88, 13, 33, 89 to 92, 14, 34, 93 to 96, 15, 35, 97 to 100, 16,36, 101 to 104, 17, 37, 105 to 108, 18, 38, 109 to 112, 19, 39, 113 to116, 20, 40, 117 to 120 or the complement thereof, or (vi) apolynucleotide encoding an amino acid sequence that, when the two aminoacid sequences are optimally aligned and compared, is at least 70%preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to theamino acid sequence encoded by the nucleotide sequence represented inany of SEQ ID NOs 277, 138, 253, 152, 198 to 201, 121, 122, 141, 154 to157, 273, 123, 142, 158 to 161, 274, 124, 143, 162 to 165, 125 to 129,144, 166 to 169, 130, 145, 170 to 173, 275, 131, 146, 174 to 177, 132,133, 147, 178 to 181, 134, 148, 182 to 185, 135, 149, 186 to 189, 136,150, 190 to 193, 276, 137, 151, 194 to 197, 139, 140, 153, 202 to 205,278, 251, 254, 257 to 260, 279, 252, 255, 256, 261 to 268, 280, 1, 21,41 to 44, 2, 22, 45 to 48, 3, 23, 49 to 52, 4, 24, 53 to 56, 5, 25, 57to 60, 6, 26, 61 to 64, 7, 27, 65 to 68, 8, 28, 69 to 72, 9, 29, 73 to76, 10, 30, 77 to 80, 11, 31, 81 to 84, 12, 32, 85 to 88, 13, 33, 89 to92, 14, 34, 93 to 96, 15, 35, 97 to 100, 16, 36, 101 to 104, 17, 37, 105to 108, 18, 38, 109 to 112, 19, 39, 113 to 116, 20, 40, 117 to 120, or(vii) a polynucleotide encoding an amino acid sequence that, when thetwo amino acid sequences are optimally aligned and compared, is at least70% preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical tothe amino acid sequence represented in any of SEQ ID NOs 285, 242, 271,226, 227, 281, 228, 282, 229, 230 to 233, 234, 283, 235, 236, 237, 238,239, 240, 284, 241, 243, 244, 286, 269, 270, 287, 288, 206 to 225. In amore preferred embodiment of the above, said polynucleotide is no longerthan 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000 or 1500nucleotides.

Preferably, the interfering RNA molecules of the current inventioncomprise at least one double-stranded region, typically the silencingelement of the interfering RNA, comprising a sense RNA strand annealedby complementary basepairing to an antisense RNA strand wherein thesense strand of the dsRNA molecule comprises a sequence of nucleotidescomplementary to a sequence of nucleotides located within the RNAtranscript of the target gene.

The silencing element, or at least one strand thereof wherein thesilencing element is double-stranded, may be fully complementary orpartially complementary to the target nucleotide sequence of the targetgene. As used herein, the term “fully complementary” means that all thebases of the nucleotide sequence of the silencing element arecomplementary to or ‘match’ the bases of the target nucleotide sequence.The term “at least partially complementary” means that there is lessthan a 100% match between the bases of the silencing element and thebases of the target nucleotide sequence. The skilled person willunderstand that the silencing element need only be at least partiallycomplementary to the target nucleotide sequence in order to mediatedown-regulation of expression of the target gene. It is known in the artthat RNA sequences with insertions, deletions and mismatches relative tothe target sequence can still be effective at RNAi. According to thecurrent invention, it is preferred that the silencing element and thetarget nucleotide sequence of the target gene share at least 80% or 85%sequence identity, preferably at least 90% or 95% sequence identity, ormore preferably at least 97% or 98% sequence identity and still morepreferably at least 99% sequence identity. Alternatively, the silencingelement may comprise 1, 2 or 3 mismatches as compared with the targetnucleotide sequence over every length of 24 partially complementarynucleotides.

It will be appreciated by the person skilled in the art that the degreeof complementarity shared between the silencing element and the targetnucleotide sequence may vary depending on the target gene to bedown-regulated or depending on the insect pest species in which geneexpression is to be controlled.

In another embodiment of the current invention, the silencing elementcomprises a sequence of nucleotides that is the RNA equivalent of any ofthe polynucleotides selected from the group consisting of apolynucleotide which comprises at least 21, preferably at least 22, 23,24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100,110, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450, 500, 550, 600,700, 800, 900, 1000, 1100 or 1115 contiguous nucleotides of a nucleotidesequence as represented by any of SEQ ID NOs 277, 138, 253, 152, 198 to201, 121, 122, 141, 154 to 157, 273, 123, 142, 158 to 161, 274, 124,143, 162 to 165, 125 to 129, 144, 166 to 169, 130, 145, 170 to 173, 275,131, 146, 174 to 177, 132, 133, 147, 178 to 181, 134, 148, 182 to 185,135, 149, 186 to 189, 136, 150, 190 to 193, 276, 137, 151, 194 to 197,139, 140, 153, 202 to 205, 278, 251, 254, 257 to 260, 279, 252, 255,256, 261 to 268, 280, 1, 21, 41 to 44, 2, 22, 45 to 48, 3, 23, 49 to 52,4, 24, 53 to 56, 5, 25, 57 to 60, 6, 26, 61 to 64, 7, 27, 65 to 68, 8,28, 69 to 72, 9, 29, 73 to 76, 10, 30, 77 to 80, 11, 31, 81 to 84, 12,32, 85 to 88, 13, 33, 89 to 92, 14, 34, 93 to 96, 15, 35, 97 to 100, 16,36, 101 to 104, 17, 37, 105 to 108, 18, 38, 109 to 112, 19, 39, 113 to116, 20, 40, 117 to 120, or the complement thereof, or (ii) apolynucleotide which comprises at least 21, preferably at least 22, 23or 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100,110, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450, 500, 550, 600,700, 800, 900, 1000, 1100 or 1115 contiguous nucleotides of a nucleotidesequence as represented in any of SEQ ID NOs 277, 138, 253, 152, 198 to201, 121, 122, 141, 154 to 157, 273, 123, 142, 158 to 161, 274, 124,143, 162 to 165, 125 to 129, 144, 166 to 169, 130, 145, 170 to 173, 275,131, 146, 174 to 177, 132, 133, 147, 178 to 181, 134, 148, 182 to 185,135, 149, 186 to 189, 136, 150, 190 to 193, 276, 137, 151, 194 to 197,139, 140, 153, 202 to 205, 278, 251, 254, 257 to 260, 279, 252, 255,256, 261 to 268, 280, 1, 21, 41 to 44, 2, 22, 45 to 48, 3, 23, 49 to 52,4, 24, 53 to 56, 5, 25, 57 to 60, 6, 26, 61 to 64, 7, 27, 65 to 68, 8,28, 69 to 72, 9, 29, 73 to 76, 10, 30, 77 to 80, 11, 31, 81 to 84, 12,32, 85 to 88, 13, 33, 89 to 92, 14, 34, 93 to 96, 15, 35, 97 to 100, 16,36, 101 to 104, 17, 37, 105 to 108, 18, 38, 109 to 112, 19, 39, 113 to116, 20, 40, 117 to 120, or the complement thereof, that, when the twosequences are optimally aligned and compared, said polynucleotide is atleast 75% preferably at least 80%, 85%, 90%, 95%, 98% or 99% identicalto any of SEQ ID NOs 277, 138, 253, 152, 198 to 201, 121, 122, 141, 154to 157, 273, 123, 142, 158 to 161, 274, 124, 143, 162 to 165, 125 to129, 144, 166 to 169, 130, 145, 170 to 173, 275, 131, 146, 174 to 177,132, 133, 147, 178 to 181, 134, 148, 182 to 185, 135, 149, 186 to 189,136, 150, 190 to 193, 276, 137, 151, 194 to 197, 139, 140, 153, 202 to205, 278, 251, 254, 257 to 260, 279, 252, 255, 256, 261 to 268, 280, 1,21, 41 to 44, 2, 22, 45 to 48, 3, 23, 49 to 52, 4, 24, 53 to 56, 5, 25,57 to 60, 6, 26, 61 to 64, 7, 27, 65 to 68, 8, 28, 69 to 72, 9, 29, 73to 76, 10, 30, 77 to 80, 11, 31, 81 to 84, 12, 32, 85 to 88, 13, 33, 89to 92, 14, 34, 93 to 96, 15, 35, 97 to 100, 16, 36, 101 to 104, 17, 37,105 to 108, 18, 38, 109 to 112, 19, 39, 113 to 116, 20, 40, 117 to 120,or the complement thereof, or (iii) a polynucleotide which comprises afragment of at least 21, preferably at least 22, 23 or 24, 25, 26, 27,28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 125, 150, 175,200, 225, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, 1000,1100 or 1115 contiguous nucleotides of a nucleotide as represented inany of SEQ ID NOs 277, 138, 253, 152, 198 to 201, 121, 122, 141, 154 to157, 273, 123, 142, 158 to 161, 274, 124, 143, 162 to 165, 125 to 129,144, 166 to 169, 130, 145, 170 to 173, 275, 131, 146, 174 to 177, 132,133, 147, 178 to 181, 134, 148, 182 to 185, 135, 149, 186 to 189, 136,150, 190 to 193, 276, 137, 151, 194 to 197, 139, 140, 153, 202 to 205,278, 251, 254, 257 to 260, 279, 252, 255, 256, 261 to 268, 280, 1, 21,41 to 44, 2, 22, 45 to 48, 3, 23, 49 to 52, 4, 24, 53 to 56, 5, 25, 57to 60, 6, 26, 61 to 64, 7, 27, 65 to 68, 8, 28, 69 to 72, 9, 29, 73 to76, 10, 30, 77 to 80, 11, 31, 81 to 84, 12, 32, 85 to 88, 13, 33, 89 to92, 14, 34, 93 to 96, 15, 35, 97 to 100, 16, 36, 101 to 104, 17, 37, 105to 108, 18, 38, 109 to 112, 19, 39, 113 to 116, 20, 40, 117 to 120, orthe complement thereof, and wherein said fragment or said complement hasa nucleotide sequence that, when said fragment is optimally aligned andcompared with the corresponding fragment in any of SEQ ID NOs 277, 138,253, 152, 198 to 201, 121, 122, 141, 154 to 157, 273, 123, 142, 158 to161, 274, 124, 143, 162 to 165, 125 to 129, 144, 166 to 169, 130, 145,170 to 173, 275, 131, 146, 174 to 177, 132, 133, 147, 178 to 181, 134,148, 182 to 185, 135, 149, 186 to 189, 136, 150, 190 to 193, 276, 137,151, 194 to 197, 139, 140, 153, 202 to 205, 278, 251, 254, 257 to 260,279, 252, 255, 256, 261 to 268, 280, 1, 21, 41 to 44, 2, 22, 45 to 48,3, 23, 49 to 52, 4, 24, 53 to 56, 5, 25, 57 to 60, 6, 26, 61 to 64, 7,27, 65 to 68, 8, 28, 69 to 72, 9, 29, 73 to 76, 10, 30, 77 to 80, 11,31, 81 to 84, 12, 32, 85 to 88, 13, 33, 89 to 92, 14, 34, 93 to 96, 15,35, 97 to 100, 16, 36, 101 to 104, 17, 37, 105 to 108, 18, 38, 109 to112, 19, 39, 113 to 116, 20, 40, 117 to 120, said nucleotide sequence isat least 75% preferably at least 80%, 85%, 90%, 95%, 98% or 99%identical to said corresponding fragment of any of SEQ ID NOs 277, 138,253, 152, 198 to 201, 121, 122, 141, 154 to 157, 273, 123, 142, 158 to161, 274, 124, 143, 162 to 165, 125 to 129, 144, 166 to 169, 130, 145,170 to 173, 275, 131, 146, 174 to 177, 132, 133, 147, 178 to 181, 134,148, 182 to 185, 135, 149, 186 to 189, 136, 150, 190 to 193, 276, 137,151, 194 to 197, 139, 140, 153, 202 to 205, 278, 251, 254, 257 to 260,279, 252, 255, 256, 261 to 268, 280, 1, 21, 41 to 44, 2, 22, 45 to 48,3, 23, 49 to 52, 4, 24, 53 to 56, 5, 25, 57 to 60, 6, 26, 61 to 64, 7,27, 65 to 68, 8, 28, 69 to 72, 9, 29, 73 to 76, 10, 30, 77 to 80, 11,31, 81 to 84, 12, 32, 85 to 88, 13, 33, 89 to 92, 14, 34, 93 to 96, 15,35, 97 to 100, 16, 36, 101 to 104, 17, 37, 105 to 108, 18, 38, 109 to112, 19, 39, 113 to 116, 20, 40, 117 to 120 or the complement thereof,wherein said polynucleotide is no longer than 10000, 9000, 8000, 7000,6000, 5000, 4000, 3000, 2000 or 1500 nucleotides. It will be appreciatedthat in such embodiments the silencing element may comprise or consistof a region of double-stranded RNA comprising annealed complementarystrands, one strand of which, the sense strand, comprises a sequence ofnucleotides at least partially complementary to a target nucleotidesequence within a target gene.

The target nucleotide sequence may be selected from any suitable regionor nucleotide sequence of the target gene or RNA transcript thereof. Forexample, the target nucleotide sequence may be located within the 5′UTRor 3′UTR of the target gene or RNA transcript or within exonic orintronic regions of the gene.

The skilled person will be aware of methods of identifying the mostsuitable target nucleotide sequences within the context of thefull-length target gene. For example, multiple silencing elementstargeting different regions of the target gene can be synthesised andtested. Alternatively, digestion of the RNA transcript with enzymes suchas RNAse H can be used to determine sites on the RNA that are in aconformation susceptible to gene silencing. Target sites may also beidentified using in silico approaches, for example, the use of computeralgorithms designed to predict the efficacy of gene silencing based ontargeting different sites within the full-length gene.

The interfering RNAs of the current invention may comprise one silencingelement or multiple silencing elements, wherein each silencing elementcomprises or consists of a sequence of nucleotides which is at leastpartially complementary to a target nucleotide sequence within a targetgene and that functions upon uptake by an insect pest species todown-regulate expression of said target gene. Concatemeric RNAconstructs of this type are described in WO2006/046148 as incorporatedherein by reference. In the context of the present invention, the term‘multiple’ means at least two, at least three, at least four, etc and upto at least 10, 15, 20 or at least 30. In one embodiment, theinterfering RNA comprises multiple copies of a single silencing elementi.e. repeats of a silencing element that binds to a particular targetnucleotide sequence within a specific target gene. In anotherembodiment, the silencing elements within the interfering RNA compriseor consist of different sequences of nucleotides complementary todifferent target nucleotide sequences. It should be clear thatcombinations of multiple copies of the same silencing element combinedwith silencing elements binding to different target nucleotide sequencesare within the scope of the current invention.

The different target nucleotide sequences may originate from a singletarget gene in an insect pest species in order to achieve improveddown-regulation of a specific target gene in an insect pest species. Inthis case, the silencing elements may be combined in the interfering RNAin the original order in which the target nucleotide sequences occur inthe target gene, or the silencing elements may be scrambled and combinedrandomly in any rank order in the context of the interfering RNA ascompared with the order of the target nucleotide sequences in the targetgene.

Alternatively, the different target nucleotide sequences arerepresenting a single target gene but originating from different insectpest species.

Alternatively, the different target nucleotide sequences may originatefrom different target genes. If the interfering RNA is for use inpreventing and/or controlling pest infestation, it is preferred that thedifferent target genes are chosen from the group of genes regulatingessential biological functions of insect pest species, including but notlimited to survival, growth, development, reproduction andpathogenicity. The target genes may regulate the same or differentbiological pathways or processes. In one embodiment, at least one of thesilencing elements comprises or consists of a sequence of nucleotideswhich is at least partially complementary to a target nucleotidesequence within a target gene wherein the target gene is selected fromthe group of genes having a nucleotide sequence comprising any of SEQ IDNOs 277, 138, 253, 152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125to 129, 144, 130, 145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149,136, 150, 276, 137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255,256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29,10, 30, 11, 31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38,19, 39, 20 or 40, or the complement thereof, or having a nucleotidesequence that, when the two sequences are optimally aligned andcompared, is at least 75%, preferably at least 80%, 85%, 90%, 95%, 98%or 99% identical to any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141,273, 123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146,132, 133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140,153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24,5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14,34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, or the complementthereof, or (ii) is selected from the group of genes having a nucleotidesequence comprising a fragment of at least 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 125, 150,175, 200, 225, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 9001000, 1100 or 1115 contiguous nucleotides of any of SEQ ID NOs 277, 138,253, 152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144,130, 145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150,276, 137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1,21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11,31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20or 40, or the complement thereof, or having a nucleotide sequence that,when said gene comprising said fragment is optimally aligned andcompared with any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273,123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146, 132,133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140, 153,278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5,25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34,15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, said nucleotidesequence is at least 75% preferably at least 80%, 85%, 90%, 95%, 98% or99% identical to any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141,273, 123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146,132, 133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140,153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24,5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14,34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, or the complementthereof, or (iii) is selected from the group of genes having anucleotide sequence comprising a fragment of at least 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100,110, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450, 500, 550, 600,700, 800, 900 1000, 1100 or 1115 contiguous nucleotides of any of SEQ IDNOs 277, 138, 253, 152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125to 129, 144, 130, 145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149,136, 150, 276, 137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255,256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29,10, 30, 11, 31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38,19, 39, 20 or 40, or the complement thereof, and wherein when saidfragment is optimally aligned and compared with the correspondingfragment in any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273,123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146, 132,133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140, 153,278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5,25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34,15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, said nucleotidesequence of said fragment is at least 75% preferably at least 80%, 85%,90%, 95%, 98% or 99% identical to said corresponding fragment of any ofSEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273, 123, 142, 274, 124,143, 125 to 129, 144, 130, 145, 275, 131, 146, 132, 133, 147, 134, 148,135, 149, 136, 150, 276, 137, 151, 139, 140, 153, 278, 251, 254, 279,252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8,28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17,37, 18, 38, 19, 39, 20 or 40, or the complement thereof, or (iv) is aninsect pest orthologue of a gene having a nucleotide sequence comprisingany of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273, 123, 142, 274,124, 143, 125 to 129, 144, 130, 145, 275, 131, 146, 132, 133, 147, 134,148, 135, 149, 136, 150, 276, 137, 151, 139, 140, 153, 278, 251, 254,279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7,27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34, 15, 35, 16,36, 17, 37, 18, 38, 19, 39, 20 or 40, or the complement thereof, whereinthe two orthologous genes are similar in sequence to such a degree thatwhen the two genes are optimally aligned and compared, the orthologuehas a sequence that is at least 75% preferably at least 80%, 85%, 90%,95%, 98% or 99% identical to any of the sequences represented by SEQ IDNOs 277, 138, 253, 152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125to 129, 144, 130, 145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149,136, 150, 276, 137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255,256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29,10, 30, 11, 31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38,19, 39, 20 or 40, or (v) is selected from the group of genes having anucleotide sequence encoding an amino acid sequence that, when the twoamino acid sequences are optimally aligned and compared, is at least 70%preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to theamino acid sequence encoded by the nucleotide sequence represented inany of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273, 123, 142, 274,124, 143, 125 to 129, 144, 130, 145, 275, 131, 146, 132, 133, 147, 134,148, 135, 149, 136, 150, 276, 137, 151, 139, 140, 153, 278, 251, 254,279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7,27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34, 15, 35, 16,36, 17, 37, 18, 38, 19, 39, 20 or 40, or (vi) is selected from the groupof genes having a nucleotide sequence encoding an amino acid sequencethat, when the two amino acid sequences are optimally aligned andcompared, is at least 70% preferably at least 75%, 80%, 85%, 90%, 95%,98% or 99% identical to the amino acid sequence represented in any ofSEQ ID NOs 285, 242, 271, 226, 227, 281, 228, 282, 229, 230 to 233, 234,283, 235, 236, 237, 238, 239, 240, 284, 241, 243, 244, 286, 269, 270,287, 288, 206 to 225. Preferably, the nucleotide sequence of the targetgene is no longer than 5000, 4000, 3000, 2000 or 1500 nucleotides.

In a further embodiment of the invention, the different genes targetedby the different silencing elements originate from the same insect pestspecies. This approach is designed to achieve enhanced attack against asingle insect pest species. In particular, the different target genesmay be expressed differentially in the different stages of the insect'slife cycle, for example, the mature adult, immature larval and eggstages. The interfering RNA of the invention may thus be used to preventand/or control insect pest infestation at more than one stage of theinsect's life cycle.

In an alternative embodiment of the invention, the different genestargeted by the different silencing elements originate from differentinsect pest species. The interfering RNA of the invention can thus beused to prevent and/or control infestation by more than one insect pestspecies simultaneously.

The silencing elements may be arranged as one contiguous region of theinterfering RNA or may be separated by the presence of linker sequences.The linker sequence may comprise a short random nucleotide sequence thatis not complementary to any target nucleotide sequences or target genes.In one embodiment, the linker is a conditionally self-cleaving RNAsequence, preferably a pH-sensitive linker or a hydrophobic-sensitivelinker. In one embodiment, the linker comprises a sequence ofnucleotides equivalent to an intronic sequence. Linker sequences of thecurrent invention may range in length from about 1 base pair to about10,000 base pairs, provided that the linker does not impair the abilityof the interfering RNA to down-regulate the expression of targetgene(s).

In addition to the silencing element(s) and any linker sequences, theinterfering RNA of the invention may comprise at least one additionalpolynucleotide sequence. In different embodiments of the invention, theadditional sequence is chosen from (i) a sequence capable of protectingthe interfering RNA against RNA processing, (ii) a sequence affectingthe stability of the interfering RNA, (iii) a sequence allowing proteinbinding, for example to facilitate uptake of the interfering RNA bycells of the insect pest species, (iv) a sequence facilitatinglarge-scale production of the interfering RNA, (v) a sequence which isan aptamer that binds to a receptor or to a molecule on the surface ofthe insect pest cells to facilitate uptake, or (v) a sequence thatcatalyses processing of the interfering RNA within the insect pest cellsand thereby enhances the efficacy of the interfering RNA. Structures forenhancing the stability of RNA molecules are well known in the art andare described further in WO2006/046148 as incorporated herein byreference.

The length of the interfering RNA of the invention needs to besufficient for uptake by the cells of an insect pest species anddown-regulation of target genes within the pest as described elsewhereherein. However, the upper limit on length may be dependent on (i) therequirement for the interfering RNA to be taken up by cells of the pestand (ii) the requirement for the interfering RNA to be processed in thecells of the pest to mediate gene silencing via the RNAi pathway. Thelength may also be dictated by the method of production and theformulation for delivery of the interfering RNA to cells. Preferably,the interfering RNA of the current invention will be between 21 and10,000 nucleotides in length, preferably between 50 and 5000 nucleotidesor between 100 and 2500 nucleotides, more preferably between 80 and 2000nucleotides in length.

The interfering RNA may contain DNA bases, non-natural bases ornon-natural backbone linkages or modifications of the sugar-phosphatebackbone, for example to enhance stability during storage or enhanceresistance to degradation by nucleases. Furthermore, the interfering RNAmay be produced chemically or enzymatically by one skilled in the artthrough manual or automated reactions.

Alternatively, the interfering RNA may be transcribed from apolynucleotide encoding the same. Thus, provided herein is an isolatedpolynucleotide encoding any of the interfering RNAs of the currentinvention.

Also provided herein is an isolated polynucleotide selected from thegroup consisting of (i) a polynucleotide which comprises at least 21,preferably at least 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50,55, 60, 70, 80, 90, 100, 110, 125, 150, 175, 200, 225, 250, 300, 350,400, 450, 500, 550, 600, 700, 800, 900, 1000, 1100 or 1115 contiguousnucleotides of a nucleotide sequence as represented by any of SEQ ID NOs277, 138, 253, 152, 198 to 201, 121, 122, 141, 154 to 157, 273, 123,142, 158 to 161, 274, 124, 143, 162 to 165, 125 to 129, 144, 166 to 169,130, 145, 170 to 173, 275, 131, 146, 174 to 177, 132, 133, 147, 178 to181, 134, 148, 182 to 185, 135, 149, 186 to 189, 136, 150, 190 to 193,276, 137, 151, 194 to 197, 139, 140, 153, 202 to 205, 278, 251, 254, 257to 260, 279, 252, 255, 256, 261 to 268, 280, 1, 21, 41 to 44, 2, 22, 45to 48, 3, 23, 49 to 52, 4, 24, 53 to 56, 5, 25, 57 to 60, 6, 26, 61 to64, 7, 27, 65 to 68, 8, 28, 69 to 72, 9, 29, 73 to 76, 10, 30, 77 to 80,11, 31, 81 to 84, 12, 32, 85 to 88, 13, 33, 89 to 92, 14, 34, 93 to 96,15, 35, 97 to 100, 16, 36, 101 to 104, 17, 37, 105 to 108, 18, 38, 109to 112, 19, 39, 113 to 116, 20, 40, 117 to 120, or the complementthereof, or (ii) a polynucleotide which consists of at least 21,preferably at least 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50,55, 60, 70, 80, 90, 100, 110, 125, 150, 175, 200, 225, 250, 300, 350,400, 450, 500, 550, 600, 700, 800, 900, 1000, 1100 or 1115 contiguousnucleotides of a nucleotide sequence as represented by any of SEQ ID NOs277, 138, 253, 152, 198 to 201, 121, 122, 141, 154 to 157, 273, 123,142, 158 to 161, 274, 124, 143, 162 to 165, 125 to 129, 144, 166 to 169,130, 145, 170 to 173, 275, 131, 146, 174 to 177, 132, 133, 147, 178 to181, 134, 148, 182 to 185, 135, 149, 186 to 189, 136, 150, 190 to 193,276, 137, 151, 194 to 197, 139, 140, 153, 202 to 205, 278, 251, 254, 257to 260, 279, 252, 255, 256, 261 to 268, 280, 1, 21, 41 to 44, 2, 22, 45to 48, 3, 23, 49 to 52, 4, 24, 53 to 56, 5, 25, 57 to 60, 6, 26, 61 to64, 7, 27, 65 to 68, 8, 28, 69 to 72, 9, 29, 73 to 76, 10, 30, 77 to 80,11, 31, 81 to 84, 12, 32, 85 to 88, 13, 33, 89 to 92, 14, 34, 93 to 96,15, 35, 97 to 100, 16, 36, 101 to 104, 17, 37, 105 to 108, 18, 38, 109to 112, 19, 39, 113 to 116, 20, 40, 117 to 120, or the complementthereof, or (iii) a polynucleotide which comprises at least 21,preferably at least 22, 23 or 24, 25, 26, 27, 28, 29, 30, 35, 40, 45,50, 55, 60, 70, 80, 90, 100, 110, 125, 150, 175, 200, 225, 250, 300,350, 400, 450, 500, 550, 600, 700, 800, 900, 1000, 1100 or 1115contiguous nucleotides of a nucleotide sequence as represented in any ofSEQ ID NOs 277, 138, 253, 152, 198 to 201, 121, 122, 141, 154 to 157,273, 123, 142, 158 to 161, 274, 124, 143, 162 to 165, 125 to 129, 144,166 to 169, 130, 145, 170 to 173, 275, 131, 146, 174 to 177, 132, 133,147, 178 to 181, 134, 148, 182 to 185, 135, 149, 186 to 189, 136, 150,190 to 193, 276, 137, 151, 194 to 197, 139, 140, 153, 202 to 205, 278,251, 254, 257 to 260, 279, 252, 255, 256, 261 to 268, 280, 1, 21, 41 to44, 2, 22, 45 to 48, 3, 23, 49 to 52, 4, 24, 53 to 56, 5, 25, 57 to 60,6, 26, 61 to 64, 7, 27, 65 to 68, 8, 28, 69 to 72, 9, 29, 73 to 76, 10,30, 77 to 80, 11, 31, 81 to 84, 12, 32, 85 to 88, 13, 33, 89 to 92, 14,34, 93 to 96, 15, 35, 97 to 100, 16, 36, 101 to 104, 17, 37, 105 to 108,18, 38, 109 to 112, 19, 39, 113 to 116, 20, 40, 117 to 120, or thecomplement thereof, that, when the two sequences are optimally alignedand compared, said polynucleotide is at least 75% preferably at least80%, 85%, 90%, 95%, 98% or 99% identical to any of SEQ ID NOs 277, 138,253, 152, 198 to 201, 121, 122, 141, 154 to 157, 273, 123, 142, 158 to161, 274, 124, 143, 162 to 165, 125 to 129, 144, 166 to 169, 130, 145,170 to 173, 275, 131, 146, 174 to 177, 132, 133, 147, 178 to 181, 134,148, 182 to 185, 135, 149, 186 to 189, 136, 150, 190 to 193, 276, 137,151, 194 to 197, 139, 140, 153, 202 to 205, 278, 251, 254, 257 to 260,279, 252, 255, 256, 261 to 268, 280, 1, 21, 41 to 44, 2, 22, 45 to 48,3, 23, 49 to 52, 4, 24, 53 to 56, 5, 25, 57 to 60, 6, 26, 61 to 64, 7,27, 65 to 68, 8, 28, 69 to 72, 9, 29, 73 to 76, 10, 30, 77 to 80, 11,31, 81 to 84, 12, 32, 85 to 88, 13, 33, 89 to 92, 14, 34, 93 to 96, 15,35, 97 to 100, 16, 36, 101 to 104, 17, 37, 105 to 108, 18, 38, 109 to112, 19, 39, 113 to 116, 20, 40, 117 to 120, or the complement thereof,or (iv) a polynucleotide which comprises a fragment of at least 21,preferably at least 22, 23 or 24, 25, 26, 27, 28, 29, 30, 35, 40, 45,50, 55, 60, 70, 80, 90, 100, 110, 125, 150, 175, 200, 225, 250, 300,350, 400, 450, 500, 550, 600, 700, 800, 900, 1000, 1100 or 1115contiguous nucleotides of a nucleotide as represented in any of SEQ IDNOs 277, 138, 253, 152, 198 to 201, 121, 122, 141, 154 to 157, 273, 123,142, 158 to 161, 274, 124, 143, 162 to 165, 125 to 129, 144, 166 to 169,130, 145, 170 to 173, 275, 131, 146, 174 to 177, 132, 133, 147, 178 to181, 134, 148, 182 to 185, 135, 149, 186 to 189, 136, 150, 190 to 193,276, 137, 151, 194 to 197, 139, 140, 153, 202 to 205, 278, 251, 254, 257to 260, 279, 252, 255, 256, 261 to 268, 280, 1, 21, 41 to 44, 2, 22, 45to 48, 3, 23, 49 to 52, 4, 24, 53 to 56, 5, 25, 57 to 60, 6, 26, 61 to64, 7, 27, 65 to 68, 8, 28, 69 to 72, 9, 29, 73 to 76, 10, 30, 77 to 80,11, 31, 81 to 84, 12, 32, 85 to 88, 13, 33, 89 to 92, 14, 34, 93 to 96,15, 35, 97 to 100, 16, 36, 101 to 104, 17, 37, 105 to 108, 18, 38, 109to 112, 19, 39, 113 to 116, 20, 40, 117 to 120, or the complementthereof, and wherein said fragment or said complement has a nucleotidesequence that, when said fragment is optimally aligned and compared withthe corresponding fragment in any of SEQ ID NOs 277, 138, 253, 152, 198to 201, 121, 122, 141, 154 to 157, 273, 123, 142, 158 to 161, 274, 124,143, 162 to 165, 125 to 129, 144, 166 to 169, 130, 145, 170 to 173, 275,131, 146, 174 to 177, 132, 133, 147, 178 to 181, 134, 148, 182 to 185,135, 149, 186 to 189, 136, 150, 190 to 193, 276, 137, 151, 194 to 197,139, 140, 153, 202 to 205, 278, 251, 254, 257 to 260, 279, 252, 255,256, 261 to 268, 280, 1, 21, 41 to 44, 2, 22, 45 to 48, 3, 23, 49 to 52,4, 24, 53 to 56, 5, 25, 57 to 60, 6, 26, 61 to 64, 7, 27, 65 to 68, 8,28, 69 to 72, 9, 29, 73 to 76, 10, 30, 77 to 80, 11, 31, 81 to 84, 12,32, 85 to 88, 13, 33, 89 to 92, 14, 34, 93 to 96, 15, 35, 97 to 100, 16,36, 101 to 104, 17, 37, 105 to 108, 18, 38, 109 to 112, 19, 39, 113 to116, 20, 40, 117 to 120, said nucleotide sequence is at least 75%preferably at least 80%, 85%, 90%, 95%, 98% or 99% identical to saidcorresponding fragment of any of SEQ ID NOs 277, 138, 253, 152, 198 to201, 121, 122, 141, 154 to 157, 273, 123, 142, 158 to 161, 274, 124,143, 162 to 165, 125 to 129, 144, 166 to 169, 130, 145, 170 to 173, 275,131, 146, 174 to 177, 132, 133, 147, 178 to 181, 134, 148, 182 to 185,135, 149, 186 to 189, 136, 150, 190 to 193, 276, 137, 151, 194 to 197,139, 140, 153, 202 to 205, 278, 251, 254, 257 to 260, 279, 252, 255,256, 261 to 268, 280, 1, 21, 41 to 44, 2, 22, 45 to 48, 3, 23, 49 to 52,4, 24, 53 to 56, 5, 25, 57 to 60, 6, 26, 61 to 64, 7, 27, 65 to 68, 8,28, 69 to 72, 9, 29, 73 to 76, 10, 30, 77 to 80, 11, 31, 81 to 84, 12,32, 85 to 88, 13, 33, 89 to 92, 14, 34, 93 to 96, 15, 35, 97 to 100, 16,36, 101 to 104, 17, 37, 105 to 108, 18, 38, 109 to 112, 19, 39, 113 to116, 20, 40, 117 to 120 or the complement thereof, or (v) apolynucleotide which consists of a fragment of at least 21, preferablyat least 22, 23 or 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60,70, 80, 90, 100, 110, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450,500, 550, 600, 700, 800, 900, 1000, 1100 or 1115 contiguous nucleotidesof a nucleotide as represented in any of SEQ ID NOs 277, 138, 253, 152,198 to 201, 121, 122, 141, 154 to 157, 273, 123, 142, 158 to 161, 274,124, 143, 162 to 165, 125 to 129, 144, 166 to 169, 130, 145, 170 to 173,275, 131, 146, 174 to 177, 132, 133, 147, 178 to 181, 134, 148, 182 to185, 135, 149, 186 to 189, 136, 150, 190 to 193, 276, 137, 151, 194 to197, 139, 140, 153, 202 to 205, 278, 251, 254, 257 to 260, 279, 252,255, 256, 261 to 268, 280, 1, 21, 41 to 44, 2, 22, 45 to 48, 3, 23, 49to 52, 4, 24, 53 to 56, 5, 25, 57 to 60, 6, 26, 61 to 64, 7, 27, 65 to68, 8, 28, 69 to 72, 9, 29, 73 to 76, 10, 30, 77 to 80, 11, 31, 81 to84, 12, 32, 85 to 88, 13, 33, 89 to 92, 14, 34, 93 to 96, 15, 35, 97 to100, 16, 36, 101 to 104, 17, 37, 105 to 108, 18, 38, 109 to 112, 19, 39,113 to 116, 20, 40, 117 to 120, or the complement thereof, and whereinsaid fragment or said complement has a nucleotide sequence that, whensaid fragment is optimally aligned and compared with the correspondingfragment in any of SEQ ID NOs 277, 138, 253, 152, 198 to 201, 121, 122,141, 154 to 157, 273, 123, 142, 158 to 161, 274, 124, 143, 162 to 165,125 to 129, 144, 166 to 169, 130, 145, 170 to 173, 275, 131, 146, 174 to177, 132, 133, 147, 178 to 181, 134, 148, 182 to 185, 135, 149, 186 to189, 136, 150, 190 to 193, 276, 137, 151, 194 to 197, 139, 140, 153, 202to 205, 278, 251, 254, 257 to 260, 279, 252, 255, 256, 261 to 268, 280,1, 21, 41 to 44, 2, 22, 45 to 48, 3, 23, 49 to 52, 4, 24, 53 to 56, 5,25, 57 to 60, 6, 26, 61 to 64, 7, 27, 65 to 68, 8, 28, 69 to 72, 9, 29,73 to 76, 10, 30, 77 to 80, 11, 31, 81 to 84, 12, 32, 85 to 88, 13, 33,89 to 92, 14, 34, 93 to 96, 15, 35, 97 to 100, 16, 36, 101 to 104, 17,37, 105 to 108, 18, 38, 109 to 112, 19, 39, 113 to 116, 20, 40, 117 to120, said nucleotide sequence is at least 75% preferably at least 80%,85%, 90%, 95%, 98% or 99% identical to said corresponding fragment ofany of SEQ ID NOs 277, 138, 253, 152, 198 to 201, 121, 122, 141, 154 to157, 273, 123, 142, 158 to 161, 274, 124, 143, 162 to 165, 125 to 129,144, 166 to 169, 130, 145, 170 to 173, 275, 131, 146, 174 to 177, 132,133, 147, 178 to 181, 134, 148, 182 to 185, 135, 149, 186 to 189, 136,150, 190 to 193, 276, 137, 151, 194 to 197, 139, 140, 153, 202 to 205,278, 251, 254, 257 to 260, 279, 252, 255, 256, 261 to 268, 280, 1, 21,41 to 44, 2, 22, 45 to 48, 3, 23, 49 to 52, 4, 24, 53 to 56, 5, 25, 57to 60, 6, 26, 61 to 64, 7, 27, 65 to 68, 8, 28, 69 to 72, 9, 29, 73 to76, 10, 30, 77 to 80, 11, 31, 81 to 84, 12, 32, 85 to 88, 13, 33, 89 to92, 14, 34, 93 to 96, 15, 35, 97 to 100, 16, 36, 101 to 104, 17, 37, 105to 108, 18, 38, 109 to 112, 19, 39, 113 to 116, 20, 40, 117 to 120 orthe complement thereof, or (vi) a polynucleotide encoding an amino acidsequence that, when the two amino acid sequences are optimally alignedand compared, is at least 70% preferably at least 75%, 80%, 85%, 90%,95%, 98% or 99% identical to the amino acid sequence encoded by thenucleotide sequence represented in any of SEQ ID NOs 277, 138, 253, 152,198 to 201, 121, 122, 141, 154 to 157, 273, 123, 142, 158 to 161, 274,124, 143, 162 to 165, 125 to 129, 144, 166 to 169, 130, 145, 170 to 173,275, 131, 146, 174 to 177, 132, 133, 147, 178 to 181, 134, 148, 182 to185, 135, 149, 186 to 189, 136, 150, 190 to 193, 276, 137, 151, 194 to197, 139, 140, 153, 202 to 205, 278, 251, 254, 257 to 260, 279, 252,255, 256, 261 to 268, 280, 1, 21, 41 to 44, 2, 22, 45 to 48, 3, 23, 49to 52, 4, 24, 53 to 56, 5, 25, 57 to 60, 6, 26, 61 to 64, 7, 27, 65 to68, 8, 28, 69 to 72, 9, 29, 73 to 76, 10, 30, 77 to 80, 11, 31, 81 to84, 12, 32, 85 to 88, 13, 33, 89 to 92, 14, 34, 93 to 96, 15, 35, 97 to100, 16, 36, 101 to 104, 17, 37, 105 to 108, 18, 38, 109 to 112, 19, 39,113 to 116, 20, 40, 117 to 120, or (vii) a polynucleotide encoding anamino acid sequence that, when the two amino acid sequences areoptimally aligned and compared, is at least 70% preferably at least 75%,80%, 85%, 90%, 95%, 98% or 99% identical to the amino acid sequencerepresented in any of SEQ ID NOs 285, 242, 271, 226, 227, 281, 228, 282,229, 230 to 233, 234, 283, 235, 236, 237, 238, 239, 240, 284, 241, 243,244, 286, 269, 270, 287, 288, 206 to 225, and wherein saidpolynucleotide is no longer than 10000, 9000, 8000, 7000, 6000, 5000,4000, 3000, 2000 or 1500 nucleotides.

In preferred embodiments, the isolated polynucleotide is part of aninterfering RNA molecule, typically part of the silencing element,comprising at least one double-stranded region comprising a sense RNAstrand annealed by complementary basepairing to an antisense RNA strandwherein the sense strand of the dsRNA molecule comprises a sequence ofnucleotides complementary to a sequence of nucleotides located withinthe RNA transcript of the target gene. The sense strand of the dsRNA istherefore able to anneal to the RNA transcript and target the RNA fordegradation within the RNAi-induced silencing complex or RISC.

The polynucleotides of the invention may be inserted via routinemolecular cloning techniques into DNA constructs or vectors known in theart. Therefore, according to one embodiment, a DNA construct comprisingany of the polynucleotides of the current invention is provided.Preferably, provided herein is a DNA construct comprising apolynucleotide encoding any of the interfering RNAs of the currentinvention. The DNA construct may be a recombinant DNA vector, forexample a bacterial or yeast vector or plasmid. In a preferredembodiment of the invention, the DNA construct is an expressionconstruct and the polynucleotide is operably linked to at least oneregulatory sequence capable of driving expression of the polynucleotidesequence. The term ‘regulatory sequence’ is to be taken in a broadcontext and is intended to refer to any nucleotide sequence capable ofeffecting expression of polynucleotides to which it is operably linkedincluding but not limited to promoters, enhancers and othernaturally-occurring or synthetic transcriptional activator elements. Theregulatory sequence may be located at the 5′ or 3′ end of thepolynucleotide sequence. The term ‘operably linked’ refers to afunctional linkage between the regulatory sequence and thepolynucleotide sequence such that the regulatory sequence drivesexpression of the polynucleotide. Operably linked elements may becontiguous or non-contiguous.

Preferably, the regulatory sequence is a promoter selected from thegroup comprising but not limited to constitutive promoters, induciblepromoters, tissue-specific promoters and growth/developmentalstage-specific promoters. In one embodiment, the polynucleotide isplaced under the control of a strong constitutive promoter such as anyselected from the group comprising the CaMV35S promoter, doubled CaMV35Spromoter, ubiquitin promoter, actin promoter, rubisco promoter, GOS2promoter, Figwort mosaic virus 34S promoter. In another embodiment, theregulatory sequence is a plant promoter for use in regulating expressionof the polynucleotide in plants. Plant promoters, in particular,tissue-specific plant promoters encompassed within the scope of thecurrent invention are described in more detail elsewhere herein.

Optionally, one or more transcription termination sequences may beincorporated in the expression construct of the invention. The term‘transcription termination sequence’ encompasses a control sequence atthe end of a transcriptional unit, which signals termination oftranscription, 3′ processing and poly-adenylation of a primarytranscript. Additional regulatory sequences including but not limited totranscriptional or translational enhancers may be incorporated in theexpression construct, for instance as with the double enhanced CaMV35Spromoter.

The present invention also encompasses a method for generating any ofthe interfering RNAs of the invention comprising the steps of (i)contacting a polynucleotide encoding said interfering RNA or a DNAconstruct comprising the same with cell-free components; or (ii)introducing (e.g. by transformation, transfection or injection) apolynucleotide encoding said interfering RNA or a DNA constructcomprising the same into a cell. Accordingly, also provided herein is ahost cell comprising any of the interfering RNAs of the currentinvention, any of the polynucleotides of the current invention or a DNAconstruct comprising the same. The host cell may be a prokaryotic cellincluding but not limited to gram-positive and gram-negative bacterialcells, or an eukaryotic cell including but not limited to yeast cells orplant cells. Preferably, said host cell is a bacterial cell or a plantcell. The bacterial cell can be chosen from the group comprising, butnot limited to, Gram positive and Gram negative cells comprisingEscherichia spp. (e.g. E. coli), Bacillus spp. (e.g. B. thuringiensis),Rhizobium spp., Lactobacilllus spp., Lactococcus spp., Pseudomonas spp.and Agrobacterium spp. The polynucleotide or DNA construct of theinvention may exist or be maintained in the host cell as anextra-chromosomal element or may be stably incorporated into the genomeof the host cell. Characteristics of particular interest in selecting ahost cell for the purposes of the current invention include the easewith which the polynucleotide or DNA construct encoding the interferingRNA can be introduced into the host, the availability of compatibleexpression systems, the efficiency of expression, and the stability ofthe interfering RNA in the host.

The DNA construct of the invention may further include an origin ofreplication which is required for maintenance and/or replication in aspecific cell type or host cell. One example is when an expressionconstruct is required to be maintained in a bacterial cell as anextra-chromosomal or episomal genetic element (e.g. a plasmid or cosmidmolecule) in a cell. Preferred origins of replication include but arenot limited to fl-ori, pBR322 ori (pMB1) and colE1 ori.

The recombinant construct may optionally comprise a selectable markergene. As used herein, the term ‘selectable marker gene’ includes anygene, which confers a phenotype on a cell in which it is expressed tofacilitate the identification and/or selection of cells, which aretransfected or transformed with an expression construct of theinvention. Examples of suitable selectable markers include resistancegenes against ampicillin (Ampr), tetracycline (Tcr), kanamycin (Kanr),phosphinothricin, and chloramphenicol (CAT). Other suitable marker genesprovide a metabolic trait, for example manA. Visual marker genes mayalso be used and include for example beta-glucuronidase (GUS),luciferase and Green Fluorescent Protein (GFP).

In situations wherein the interfering RNA is expressed within a hostcell and/or is used to prevent and/or control pest infestation of a hostorganism, it is preferred that the interfering RNA does not exhibitsignificant ‘off-target’ effects i.e. the interfering RNA does notaffect expression of genes within the host. Preferably, the silencingelement does not exhibit significant complementarity with nucleotidesequences other than the intended target nucleotide sequence of thetarget gene. In one embodiment of the invention, the silencing elementshows less than 30%, more preferably less than 20%, more preferably lessthan 10% and even more preferably less than 5% sequence identity withany gene of the host cell or organism. If genomic sequence data isavailable for the host organism, one can cross-check identity with thesilencing element using standard bioinformatics tools. In oneembodiment, there is no sequence identity between the silencing elementand a gene from the host cell or host organism over a region of 17, morepreferably over a region of 18 or 19 and most preferably over a regionof 20 or 21 contiguous nucleotides.

Any of the interfering RNA molecules or DNA constructs encoding theinterfering RNA molecule or host cells comprising the interfering RNAmolecule as herein described may be used for the prevention and/orcontrol of insect pest infestation. As such, the interfering RNAs or DNAconstructs or host cells comprising the same may be referred to aspesticides or insecticides. Preferably, the interfering RNA moleculesand/or DNA constructs or host cells of the present invention are used totreat plants as a means to prevent and/or control pest infestationthereof. In particular, the interfering RNA molecules and/or DNAconstructs or host cells may be provided as a kit for the purposes ofpreventing and/or controlling pest infestation, preferably pestinfestation of plants.

Furthermore, in accordance with another aspect of the invention, thereis provided herein a composition for preventing and/or controllinginsect pest infestation comprising at least one interfering ribonucleicacid (RNA) and optionally at least one suitable carrier, excipient ordiluent, wherein the interfering RNA functions upon uptake by the pestto down-regulate the expression of a target gene within said pest. Theinterfering RNA may be any of those as disclosed elsewhere herein.Preferably, the interfering RNA comprises or consists of at least onesilencing element and said silencing element is a region ofdouble-stranded RNA comprising annealed complementary strands, onestrand of which (the sense strand) comprises a sequence of nucleotideswhich is at least partially complementary to a target nucleotidesequence within a target gene. The ‘target gene’ may be any of the pesttarget genes as disclosed elsewhere herein including but not limited togenes involved in regulating pest survival, growth, development,reproduction and pathogenicity. Alternatively, the composition comprisesat least one host cell comprising at least one interfering RNA moleculeor DNA construct encoding the same and optionally at least one suitablecarrier, excipient or diluent, wherein the interfering RNA functionsupon uptake of the host cell by the insect pest to down-regulate theexpression of a target gene within said pest.

In the practical application of the invention, the composition may beused for the prevention and/or control of any insect pest belonging tothe Orders Coleoptera, Lepidoptera, Diptera, Dichyoptera, Orthoptera,Hemiptera and Siphonaptera. The composition may therefore be in anysuitable form for application to insect pests or for application tosubstrates and/or organisms, in particular plants, susceptible toinfestation by said insect pest. In one embodiment, the composition isfor use in preventing and/or controlling pest infestation of plants orpropagation or reproductive material of plants and is thus directedtowards insect pest species that infest plants. The composition of thepresent invention is particularly effective when the insect pest belongsto the category of ‘chewing’ insects that cause considerable damage toplants by eating plant tissues such as roots, leaves, flowers, buds,twigs and the like. Examples from this large insect category includebeetles and their larvae. In a preferred embodiment of the invention,the insect pest is selected from the Leptinotarsa genus. Morepreferably, the target insect pest species is Leptinotarsa decemlineata.

The composition of the present invention is also effective againstspecies of insects that pierce and/or suck the fluids from the cells andtissues of plants. Thus, in a further preferred embodiment of theinvention, the insect pest is selected from the Lygus genus. Preferably,the target insect pest species is selected from the group comprisingLygus adspersus, Lygus alashanensis, Lygus borealis, Lygus elisus, Lygusgemellatus, Lygus hesperus, Lygus lineolaris or Lygus rugulipennis. Morepreferably, the target insect pest species is Lygus hesperus. Thecomposition of the invention may be used to control insect pests at allstages of their life cycle, for example, the mature adult stage, thelarval and egg stages.

In the context of the composition of the invention, the interfering RNAmay be produced from a DNA construct, in particular an expressionconstruct as described elsewhere herein, comprising a polynucleotideencoding the same. Furthermore, the interfering RNA may be producedinside a host cell or organism engineered to express said interferingRNA from a polynucleotide encoding the same. Suitable host organisms foruse in the compositions of the current invention include but are notlimited to microorganisms that are known to colonize the environment onand/or around plants or crops of interest i.e. plants or cropssusceptible to infestation by insect pest species. Such microorganismsinclude but are not limited to those that occupy the phylloplane (thesurface of plant leaves) and/or the rhizosphere (the soil surroundingplant roots). These microorganisms are selected so as to be capable ofsuccessfully competing with any wild-type organisms present in the plantenvironment. Suitable microorganisms for use as hosts include variousspecies of bacteria, algae and fungi. It is clear that the chosenmicroorganisms must not be toxic to plants.

Host organisms that do not naturally colonize plants and/or theirenvironment are also within the scope of the current invention. In oneembodiment, the interfering RNA is fermented in a bacterial host, andthe resulting dead bacteria are processed and used as an insecticidalspray in the same manner that Bacillus thuringiensis strains have beenused as an insecticide for a spray application.

Wherein the composition of the invention is for use in preventing and/orcontrolling pest infestation of a plant, the composition can contain anagriculturally suitable carrier. Such a carrier may be any material thatthe plant to be treated can tolerate, which does not cause undue damageto the environment or other organisms therein and, which allows theinterfering RNA to remain effective against the insect pest species. Inparticular, the compositions of the invention may be formulated fordelivery to plants in accordance with routine agricultural practicesused in the bioinsecticide industry. The composition may contain furthercomponents capable of performing other functions including but notlimited to (i) enhancement or promotion of uptake of the interfering RNAby cells of the pest and (ii) stabilization of the active components ofthe composition. Specific examples of such further components containedin the composition comprising the interfering RNA, are yeast tRNA oryeast total RNA.

The compositions may be formulated for direct application or as aconcentration of a primary composition that requires dilution prior touse. Alternatively, the composition may be supplied as kit comprisingthe interfering RNA or the host cell comprising or expressing the samein one container and the suitable diluent or carrier for the RNA or hostcell in a separate container. In the practical application of theinvention, the composition may be applied to a plant or any part of aplant at any stage of the plant's development. In one embodiment, thecomposition is applied to the aerial parts of a plant, for exampleduring cultivation of plant crops in a field. In a further embodiment,the composition is applied to the seeds of a plant either while they arein storage or once they are planted in the soil. It is generallyimportant to obtain good control of pests in the early stages of plantgrowth as this is the time when the plant can be most severely damagedby pest species.

The composition may be applied to the environment of an insect pest byvarious techniques including but not limited to spraying, atomizing,dusting, scattering, pouring, coating of seeds, seed treatment,introduction into the soil, and introduction into irrigation water. Inthe treatment of plants susceptible to pest infestation, the compositionmay be delivered to the plant or part of a plant before the appearanceof the pest (for the purposes of prevention), or once signs of pestinfestation begin to appear (for the purposes of pest control).

In a further embodiment of the invention, the composition is formulatedso as to contain at least one further agronomical agent, for example aherbicide or an additional pesticide. As used herein, a ‘secondpesticide’ or ‘additional pesticide’ refers to a pesticide other thanthe first or original interfering RNA molecule of the composition.Alternatively, the composition of the invention may be delivered incombination with at least one other agronomical agent, for a example aherbicide or a second pesticide. In one embodiment, the composition isprovided in combination with a herbicide selected from any known in theart, for instance glyphosate, imidazolinone, sulphonylurea andbromoxynil. In a further embodiment, the composition is provided incombination with at least one additional pesticide. The additionalpesticide may be selected from any pesticides known in the art and/ormay comprise an interfering ribonucleic acid that functions upon uptakeby a pest to down-regulate expression of a target gene in said pestspecies. In one embodiment, the target pest is an insect pest speciesand the interfering RNA is selected from any of the interfering RNAs asdescribed herein. In a further embodiment, the additional pesticidecomprises an interfering RNA that functions to down-regulate expressionof a known gene in any target pest species, not limited to insect pests.The original interfering RNA molecule of the composition and the secondor additional pesticide(s) may target the same insect pest species ormay be intended to target different insect pest species. For example,the original interfering RNA and the second pesticide may targetdifferent species of insect pest or may target different families orclasses of pest organisms, for example, fungi or nematodes or insects.It will be apparent to one skilled in the art how to test combinationsof interfering RNA molecules and other agronomical agents forsynergistic effects. In a preferred embodiment, the composition containsa first interfering RNA molecule described elsewhere herein and one ormore additional pesticides, each toxic to the same insect pest, whereinthe one or more additional pesticides are selected from a patatin, aBacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidalprotein, a Photorhabdus insecticidal protein, a Bacillus laterosporousinsecticidal protein, a Bacillus spaericus insecticidal protein, and alignin, and wherein said Bacillus thuringiensis insecticidal protein isselected from the group consisting of a Cry1Ab, a Cry1C, a Cry2Aa, aCry3, a TIC851, a CryET70, a Cry22, a VIP, a TIC901, a TIC1201, aTIC407, a TIC417, a binary insecticidal protein selected from CryET33and CryET34, CryET80 and CryET76, TIC100 and TIC101, and PS149B1, andinsecticidal chimeras of any of the preceding insecticidal proteins.

The different components of the combinations described herein may beadministered, for example to a host organism susceptible to infestationby pest, in any order. The components may be delivered simultaneously orsequentially to the area or organism to be treated.

According to a further aspect of the current invention, there isprovided herein a method for down-regulating expression of a target genein an insect pest species comprising contacting said pest with aneffective amount of at least one interfering ribonucleic acid (RNA),wherein the interfering RNA functions upon uptake by the pest todown-regulate the expression of a target gene within said pest.

The target gene may be any of the pest genes as described elsewhereherein. In particular, the target gene may be selected from the group ofgenes having a nucleotide sequence comprising any of SEQ ID NOs 277,138, 253, 152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129,144, 130, 145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136,150, 276, 137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256,280, 1, 21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10,30, 11, 31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19,39, 20 or 40, or the complement thereof, or having a nucleotide sequencethat, when the two sequences are optimally aligned and compared, is atleast 75%, preferably at least 80%, 85%, 90%, 95%, 98% or 99% identicalany of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273, 123, 142, 274,124, 143, 125 to 129, 144, 130, 145, 275, 131, 146, 132, 133, 147, 134,148, 135, 149, 136, 150, 276, 137, 151, 139, 140, 153, 278, 251, 254,279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7,27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34, 15, 35, 16,36, 17, 37, 18, 38, 19, 39, 20 or 40, or the complement thereof, or isselected from the group of genes having a nucleotide sequence consistingof any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273, 123, 142,274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146, 132, 133, 147,134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140, 153, 278, 251,254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26,7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34, 15, 35, 16,36, 17, 37, 18, 38, 19, 39, 20 or 40, or the complement thereof, or isselected from the group of genes having a nucleotide sequence comprisinga fragment of at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35,40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 125, 150, 175, 200, 225, 250,300, 350, 400, 450, 500, 550, 600, 700, 800, 900 1000, 1100 or 1115contiguous nucleotides of any of SEQ ID NOs 277, 138, 253, 152, 121,122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275,131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151,139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3,23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32,13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, or thecomplement thereof, or having a nucleotide sequence that, when said genecomprising said fragment is optimally aligned and compared with any ofSEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273, 123, 142, 274, 124,143, 125 to 129, 144, 130, 145, 275, 131, 146, 132, 133, 147, 134, 148,135, 149, 136, 150, 276, 137, 151, 139, 140, 153, 278, 251, 254, 279,252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8,28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17,37, 18, 38, 19, 39, 20 or 40, said nucleotide sequence is at least 75%preferably at least 80%, 85%, 90%, 95%, 98% or 99% identical to any ofSEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273, 123, 142, 274, 124,143, 125 to 129, 144, 130, 145, 275, 131, 146, 132, 133, 147, 134, 148,135, 149, 136, 150, 276, 137, 151, 139, 140, 153, 278, 251, 254, 279,252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8,28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17,37, 18, 38, 19, 39, 20 or 40, or the complement thereof, or is selectedfrom the group of genes having a nucleotide sequence comprising afragment of at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40,45, 50, 55, 60, 70, 80, 90, 100, 110, 125, 150, 175, 200, 225, 250, 300,350, 400, 450, 500, 550, 600, 700, 800, 900 1000, 1100 or 1115contiguous nucleotides of any of SEQ ID NOs 277, 138, 253, 152, 121,122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275,131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151,139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3,23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32,13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, or thecomplement thereof, and wherein when said fragment is optimally alignedand compared with the corresponding fragment in any of SEQ ID NOs 277,138, 253, 152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129,144, 130, 145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136,150, 276, 137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256,280, 1, 21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10,30, 11, 31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19,39, 20 or 40, said nucleotide sequence of said fragment is at least 75%preferably at least 80%, 85%, 90%, 95%, 98% or 99% identical to saidcorresponding fragment of any of SEQ ID NOs 277, 138, 253, 152, 121,122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275,131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151,139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3,23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32,13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, or thecomplement thereof.

The target gene may also be an insect pest orthologue of a gene having anucleotide sequence comprising any of SEQ ID NOs 277, 138, 253, 152,121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144, 130, 145,275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150, 276, 137,151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2,22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12,32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, orthe complement thereof, wherein the two orthologous genes are similar insequence to such a degree that when the two genes are optimally alignedand compared, the orthologue has a sequence that is at least 75%preferably at least 80%, 85%, 90%, 95%, 98% or 99% identical to any ofthe sequences represented by SEQ ID NOs 277, 138, 253, 152, 121, 122,141, 273, 123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131,146, 132, 133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139,140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23,4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13,33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40. Preferablythe nucleotide sequence of said target gene is no longer than 5000,4000, 3000, 2000 or 1500 nucleotides.

The interfering RNA for use in the present method may be any of theinterfering RNA molecules as described elsewhere herein. In oneembodiment, the interfering RNA mediates down-regulation of geneexpression by the process of RNA interference or RNAi, and theinterfering RNA is selected from the group of regulatory RNA moleculescapable of effecting RNAi or ‘gene silencing’ including but not limitedto short interfering RNAs (siRNA), microRNAs (miRNA), double-strandedRNAs (dsRNA) and hairpin RNAs (shRNA).

In preferred embodiments, the interfering RNA molecules for use in thepresent method comprise at least one silencing element wherein thesilencing element is a region of double-stranded RNA comprising a senseRNA strand annealed by complementary basepairing to an antisense RNAstrand wherein the sense strand of the dsRNA molecule comprises asequence of nucleotides complementary to a sequence of nucleotideslocated within the RNA transcript of the target gene. The sense strandof the dsRNA is therefore able to anneal to the RNA transcript andtarget the RNA for degradation within the RNAi-induced silencing complexor RISC.

In the present method, the insect pest is treated with at least oneinterfering RNA. In one embodiment of the method, the insect pestspecies may be contacted with multiple interfering RNA molecules.Wherein the pest is contacted with multiple interfering RNAs, thedifferent RNAs may function to down-regulate the same target gene ordifferent target genes.

The interfering RNA to be delivered to the insect pest species may beformulated as a composition comprising at least one suitable carrier,excipient or diluent. Furthermore, the interfering RNA may betranscribed from a polynucleotide encoding the same or a DNA constructcomprising said polynucleotide. In one embodiment, the interfering RNAis expressed inside a host cell or organism including a prokaryotic oreukaryotic host. The host cell or organism may be a host susceptible toinfestation by an insect pest wherein the interfering RNA functions todown-regulate expression of a target gene in said pest. In a preferredembodiment, the host organism is a plant susceptible to infestation bythe targeted insect pest species.

As used in the context of the present method, the term ‘contacting’refers to any means by which the insect pest species or a cell thereofis exposed to the interfering RNA and which allows for uptake of theinterfering RNA by cells of the pest. Thus, ‘contacting’ encompasses forexample, the processes of cell transformation, microinjection andfeeding. These techniques may be carried out in respect of isolatedcells grown in vitro or cells within the intact body of the insect pestspecies. Wherein the intact insect pest is contacted with theinterfering RNA of the method, the RNA may be microinjected into anextracellular space and subsequently taken up by cells of the body bynatural processes such as endocytosis or transcytosis. In a preferredembodiment of the invention, the interfering RNA is provided to the pestin the form of or included in foodstuff to be ingested by the pest. Onceingested, the interfering RNA may pass from the insect's digestive tractinto the cells of the body by natural processes such as endocytosis ortranscytosis. In one embodiment, the insect pest is exposed to a plantthat has been treated with an interfering RNA or a compositioncomprising the same, and the interfering RNA is taken up as the pestfeeds on the plant tissue. The interfering RNA may be present on thesurface of a plant or a part thereof or may be present intracellularlyin the plant or the plant tissue eaten by the insect pest.

In the context of a method for down-regulating expression of a targetgene in an insect pest species, the phrase ‘effective amount’ should betaken to mean the quantity or concentration of interfering RNA requiredto down-regulate expression of the target gene by at least 10% or 20%,preferably by at least 33%, more preferably by at least 50%, yet morepreferably by at least 80% or 90%. In particularly preferredembodiments, an ‘effective amount’ is the quantity or concentrationrequired to down-regulate expression of the target gene by at least 60%,70% or 80%, preferably by at least 90%, more preferably by at least 95%,and most preferably by at least 99% relative to expression in theabsence of an interfering RNA or in the presence of a control RNA. Asdescribed elsewhere herein, down-regulation of gene expression can bemeasured by a reduction in the levels of either the RNA transcript orthe protein ultimately produced from the target gene. Levels of RNAand/or protein can be measured using techniques routine in the art.

Also provided herein is a method for preventing and/or controlling pestinfestation, comprising contacting an insect pest species with aneffective amount of at least one interfering RNA wherein the RNAfunctions upon uptake by said pest to down-regulate expression of anessential pest target gene. The essential target gene may be any pestgene involved in the regulation of an essential biological processrequired by the pest to initiate or maintain infestation including butnot limited to survival, growth, development, reproduction andpathogenicity. In particular, the target gene may be any of the pestgenes as described elsewhere herein. Furthermore, there is providedherein a method for preventing and/or controlling insect pestinfestation in a field of crop plants, said method comprising expressingin said plants an effective amount of an interfering RNA as describedherein.

Wherein the method is for the control of pest infestation, the phrase‘effective amount’ extends to the quantity or concentration ofinterfering RNA required to produce a phenotypic effect on the pest suchthat the numbers of pest organisms infesting a host organism are reducedand/or the amount of damage caused by the pest is reduced. In oneembodiment, the phenotypic effect is death of the pest and theinterfering RNA is used to achieve at least 20%, 30%, 40%, preferably atleast 50%, 60%, 70%, more preferably at least 80% or 90% pest mortalityas compared to control insect pests. In a further embodiment, thephenotypic effects include but are not limited to stunting of pestgrowth, cessation of feeding and reduced egg-laying. The total numbersof pest organisms infesting a host organism may thus be reduced by atleast 20%, 30%, 40%, preferably at least 50%, 60%, 70%, more preferablyat least 80% or 90% as compared with control pests. Alternatively, thedamage caused by the insect pest may be reduced by at least 20%, 30%,40%, preferably at least 50%, 60%, 70%, more preferably at least 80% or90% as compared with control insect pests. Hence, the method of theinvention can be used to achieve at least 20%, 30%, 40%, preferably atleast 50%, 60%, 70%, more preferably at least 80% or 90% pest control.As used herein ‘control pests’ are pests not contacted with anypesticidal agent, or pests contacted with an interfering RNA targeting anon-essential gene i.e. a gene not required for the initiation ormaintenance of pest infestation or pests contacted with an interferingRNA targeting a gene not found and/or not expressed in said pest.

Methods for down-regulating expression of a target gene in an insectpest species may be used to prevent and/or control pest infestation on aparticular substrate or material susceptible to infestation by saidinsect pest. In one embodiment, the method is used to treat any plantsusceptible to infestation by said pest. Plants of interest for useaccording to the methods of the current invention include but are notlimited to rice, potato, cotton, tomato, canola, soy, sunflower,sorghum, pearl millet, corn, seed crops such as alfalfa, strawberries,eggplant, pepper and tobacco.

Furthermore, there is provided herein a method for increasing the yieldof a plant crop comprising contacting said plants with an effectiveamount of an interfering RNA that functions upon uptake by an insectpest species to down-regulate expression of a target gene in said pest,wherein down-regulation of the target gene affects an essentialbiological function of the pest required for initiation and/ormaintenance of infestation, such that the damage caused to the plantcrop is reduced as compared with untreated crops.

Plants or plant crops to be treated according to the methods of thecurrent invention may be treated externally with an interfering RNA orcomposition comprising the same. For example, the interfering RNA orhost cells comprising or expressing the same may be applied to thesurface of the plant or to the plant's environment by processesincluding but not limited to spraying, atomizing, dusting, scattering,pouring, coating of seeds, seed treatment, introduction into the soiland introduction into irrigation water. In one embodiment, the plant tobe treated is engineered to express the interfering RNA intracellularlyvia transcription from a polynucleotide incorporated therein. As thepest feeds on tissues of the plant, the cells containing the interferingRNA will be broken down inside the insect's digestive tract and theinterfering RNA will thus be distributed within the insect's bodyresulting in down-regulation of target genes.

Thus, in accordance with another aspect of the present invention isprovided a method for generating a transgenic plant resistant toinfestation by an insect pest species comprising the steps of (a)transforming a plant cell with a DNA construct comprising apolynucleotide sequence encoding an interfering ribonucleic acid (RNA)that functions upon uptake by an insect pest species to down-regulateexpression of a target gene in said insect pest species, (b)regenerating a plant from the transformed plant cell; and (c) growingthe transformed plant under conditions suitable for the expression ofthe interfering RNA from the recombinant DNA construct, said plant thusbeing resistant to said pest as compared with an untransformed plant.

The interfering RNA expressed by the plant or part thereof may be any ofthose as disclosed elsewhere herein. Preferably, the interfering RNAcomprises or consists of at least one silencing element and saidsilencing element is a region of double-stranded RNA comprises annealedcomplementary strands, one strand of which (the sense strand) comprisesa sequence of nucleotides which is at least partially complementary to atarget nucleotide sequence within a target gene. Wherein part of theinterfering RNA is double-stranded, the two strands of the molecule maybe expressed from at least two separate polynucleotides or may beencoded by a single polynucleotide encoding an interfering RNA with forexample, a stem-loop structure as described elsewhere herein.

The interfering RNA expressed by the plant or part thereof may targetany of the pest genes as described elsewhere herein. In particular, thetarget gene may be selected from the group of genes having a nucleotidesequence comprising any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141,273, 123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146,132, 133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140,153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24,5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14,34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, or the complementthereof, or having a nucleotide sequence that, when the two sequencesare optimally aligned and compared, is at least 75%, preferably at least80%, 85%, 90%, 95%, 98% or 99% identical any of SEQ ID NOs 277, 138,253, 152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144,130, 145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150,276, 137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1,21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11,31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20or 40, or the complement thereof, or is selected from the group of geneshaving a nucleotide sequence consisting of any of SEQ ID NOs 277, 138,253, 152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144,130, 145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150,276, 137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1,21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11,31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20or 40, or the complement thereof, or is selected from the group of geneshaving a nucleotide sequence comprising a fragment of at least 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90,100, 110, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450, 500, 550,600, 700, 800, 900 1000, 1100 or 1115 contiguous nucleotides of any ofSEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273, 123, 142, 274, 124,143, 125 to 129, 144, 130, 145, 275, 131, 146, 132, 133, 147, 134, 148,135, 149, 136, 150, 276, 137, 151, 139, 140, 153, 278, 251, 254, 279,252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8,28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17,37, 18, 38, 19, 39, 20 or 40, or the complement thereof, or having anucleotide sequence that, when said gene comprising said fragment isoptimally aligned and compared with any of SEQ ID NOs 277, 138, 253,152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144, 130,145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150, 276,137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21,2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31,12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or40, said nucleotide sequence is at least 75% preferably at least 80%,85%, 90%, 95%, 98% or 99% identical to any of SEQ ID NOs 277, 138, 253,152, 121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144, 130,145, 275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150, 276,137, 151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21,2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31,12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or40, or the complement thereof, or is selected from the group of geneshaving a nucleotide sequence comprising a fragment of at least 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90,100, 110, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450, 500, 550,600, 700, 800, 900 1000, 1100 or 1115 contiguous nucleotides of any ofSEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273, 123, 142, 274, 124,143, 125 to 129, 144, 130, 145, 275, 131, 146, 132, 133, 147, 134, 148,135, 149, 136, 150, 276, 137, 151, 139, 140, 153, 278, 251, 254, 279,252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8,28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34, 15, 35, 16, 36, 17,37, 18, 38, 19, 39, 20 or 40, or the complement thereof, and whereinwhen said fragment is optimally aligned and compared with thecorresponding fragment in any of SEQ ID NOs 277, 138, 253, 152, 121,122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275,131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151,139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3,23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32,13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, saidnucleotide sequence of said fragment is at least 75% preferably at least80%, 85%, 90%, 95%, 98% or 99% identical to said corresponding fragmentof any of SEQ ID NOs 277, 138, 253, 152, 121, 122, 141, 273, 123, 142,274, 124, 143, 125 to 129, 144, 130, 145, 275, 131, 146, 132, 133, 147,134, 148, 135, 149, 136, 150, 276, 137, 151, 139, 140, 153, 278, 251,254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23, 4, 24, 5, 25, 6, 26,7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13, 33, 14, 34, 15, 35, 16,36, 17, 37, 18, 38, 19, 39, 20 or 40, or the complement thereof. Thetarget gene may also be an insect pest orthologue of a gene having anucleotide sequence comprising any of SEQ ID NOs 277, 138, 253, 152,121, 122, 141, 273, 123, 142, 274, 124, 143, 125 to 129, 144, 130, 145,275, 131, 146, 132, 133, 147, 134, 148, 135, 149, 136, 150, 276, 137,151, 139, 140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2,22, 3, 23, 4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12,32, 13, 33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, orthe complement thereof, wherein the two orthologous genes are similar insequence to such a degree that when the two genes are optimally alignedand compared, the orthologue has a sequence that is at least 75%preferably at least 80%, 85%, 90%, 95%, 98% or 99% identical to any ofthe sequences represented by SEQ ID NOs 277, 138, 253, 152, 121, 122,141, 273, 123, 142, 274, 124, 143, 125 to 129, 144, 130, 145, 275, 131,146, 132, 133, 147, 134, 148, 135, 149, 136, 150, 276, 137, 151, 139,140, 153, 278, 251, 254, 279, 252, 255, 256, 280, 1, 21, 2, 22, 3, 23,4, 24, 5, 25, 6, 26, 7, 27, 8, 28, 9, 29, 10, 30, 11, 31, 12, 32, 13,33, 14, 34, 15, 35, 16, 36, 17, 37, 18, 38, 19, 39, 20 or 40, Preferablythe nucleotide sequence of said target gene is no longer than 5000,4000, 3000, 2000 or 1500 nucleotides. Furthermore, it is important thatthe interfering RNA does not disrupt expression of any genes of theplant host.

As used herein, the term ‘transgenic plant’ or ‘transgenic plant cell’refers to any plant or plant cell that has been genetically engineeredor is descended from a plant that has been genetically engineered so asto carry an exogenous polynucleotide sequence. ‘Exogenous’ refers to thefact that the polynucleotide originates from outside the plant cell.Typically, the exogenous polynucleotide is non-native to the transgenicplant i.e. it is not found naturally within the genome of the plant.

As used herein, the term ‘transformation’ refers to the introduction ofexogenous polynucleotide molecules into a plant or a cell thereof.Techniques for introducing polynucleotides into plants are known in theart. In one embodiment of the current invention, the plants are ‘stablytransformed’ with a polynucleotide or DNA construct comprising the same,i.e. the polynucleotide or DNA construct introduced into the plant cellintegrates into the genome of the plant and is capable of beinginherited by the progeny thereof. Transformation protocols forintroducing polynucleotides or DNA constructs into the cells of plantsmay vary depending on the type of plant concerned. Suitabletransformation methods include but are not limited to microinjection,electroporation, Agrobacterium-mediated transformation, and ballisticparticle acceleration. Methods are also known in the art for thetargeted insertion of a polynucleotide or DNA construct at a specificlocation in the plant genome using site-specific recombination systems.

The DNA construct comprising the polynucleotide encoding the activeinterfering RNA molecule may be any vector suitable for transformationof plant cells. Suitable vectors include but are not limited tobacterial plasmids, for example the Ti plasmid of Agrobacteriumtumefaciens, and viral vector systems. The DNA construct introduced intothe cells of a plant must not be harmful or toxic to the plant and/ormust not be harmful or toxic to any organisms higher up the food chainthat feed on said plants.

In one embodiment, the DNA construct is an expression constructcomprising a polynucleotide encoding an interfering RNA operably linkedto a regulatory sequence capable of driving expression of thepolynucleotide sequence in plants such as any selected from the groupcomprising the CaMV35S promoter, doubled CaMV35S promoter, ubiquitinpromoter, actin promoter, rubisco promoter, GOS2 promoter, Figwortmosaic virus 34S promoter and the double enhanced CaMV35S promoter.Preferably, the regulatory sequence is a plant promoter selected fromthose known in the art. In some embodiments, it may be preferred thatthe plant produces interfering RNA molecules only in the parts of theplant which will come into contact with and/or are damaged by the insectpest species, for example, the aerial parts of the plant, the roots etc.This effect can be achieved through the use of tissue-specific plantpromoters including but not limited to leaf-specific promoters,root-specific promoters, stem-specific promoters, flower-specificpromoters and fruit-specific promoters known in the art. Suitableexamples of a root specific promoter are PsMTA and the Class IIIChitinase promoter. Examples of leaf- and stem-specific orphotosynthetic tissue-specific promoters that are also photoactivatedare promoters of two chlorophyll binding proteins (cab1 and cab2) fromsugar beet, ribulose-bisphosphate carboxylase (Rubisco), encoded byrbcS, A (gapA) and B (gapB) subunits of chloroplastglyceraldehyde-3-phosphate dehydrogenase, promoter of the Solanumtuberosum gene encoding the leaf and stem specific (ST-LS1) protein,stem-regulated, defense-inducible genes, such as JAS promoters,flower-specific promoters such as chalcone synthase promoter andfruit-specific promoters such as that of RJ39 from strawberry.

In other embodiments, it may be preferred that the plant producesinterfering RNA molecules only at a particular stage of its growth. Thiseffect can be achieved through the use of development-specific plantpromoters that drive expression only during certain periods of plantdevelopment. In particular, it is important to protect plants from pestinfestation during the early stages of plant growth or during flowering(for instance in case of rice) or during fructification or fruitmaturation or seed-filling, as this is the time when the plant can bemost severely damaged.

The DNA construct for use in transformation of a plant according to thepresent method may comprise more than one polynucleotide encoding aninterfering RNA molecule of the current invention. In one embodiment,the different polynucleotides may encode interfering RNA moleculestargeting different nucleotide sequences within the same target gene. Ina further embodiment, the different polynucleotides may encodeinterfering RNA molecules targeting different nucleotide sequenceswithin different target genes, wherein the different target genesoriginate from the same or different insect pest species. Wherein theDNA construct encodes more than one interfering RNA, these RNAs may beexpressed differentially within different tissues of the plant by virtueof being under the control of different tissue-specific promotersequences as described elsewhere herein. In one embodiment, the plant isengineered to express an interfering RNA in the leaves whichdown-regulates expression of a target gene in an insect that feeds onthe leaves, and to additionally express an interfering RNA in the rootswhich down-regulates expression of a target gene in an insect thatcolonizes the soil and feeds on the plant roots.

The DNA construct may also comprise at least one other polynucleotide ofinterest, for example a polynucleotide encoding an additional regulatoryRNA molecule, a polynucleotide encoding a protein toxic to insect pestspecies and/or a polynucleotide encoding a protein conferring herbicideresistance.

In accordance with the present method, a plant is regenerated from atransformed plant cell using techniques known in the art. One suchtechnique comprises enzymatic digestion of the plant cell wall toproduce a plant protoplast, which can subsequently undergo multiplerounds of cell division and differentiation to produce an adult plant.Adult plants generated in such a way can be subsequently tested forresistance to pest infestation. ‘Resistant’ as used herein should beinterpreted broadly and relates to the ability of the plant to defendagainst attack from a pest that is typically capable of inflictingdamage or loss to the plant. Resistant may either be taken to mean thatthe plant is no longer susceptible to pest infestation or that anydisease symptoms resulting from pest infestation are reduced by at leastabout 20%, preferably at least 30%, 40% or 50%, more preferably at least60%, 70% or 80% and most preferably by at least 90%. Techniques tomeasure the resistance of a plant to insect pest species are commonlyknown in the art and include but are not limited to measuring over timethe average lesion diameter, the pest biomass, and/or the overallpercentage of decayed plant tissues.

In one embodiment, the present method of producing a transgenic plantalso includes the step of generating offspring or progeny from thetransgenic plant and testing the progeny for resistance to the insectpest. Two or more generations may be produced to ensure that expressionof the resistance trait is stably maintained and inherited. Seeds mayalso be harvested from the parent transgenic plant and/or its progeny totest for resistance to an insect pest.

Also encompassed within the present invention is a method for generatingtransgenic plants resistant to infestation by an insect pest speciescomprising the steps of crossing a first transgenic plant carrying a DNAconstruct encoding an interfering RNA that functions to down-regulateexpression of a pest target gene, with a second plant lacking said DNAconstruct, and selecting progeny resistant to said pest. Crossing may becarried out by any methods routinely used in the context of plantbreeding. The progeny selected for pest resistance may additionally beself-pollinated or ‘selfed’ to produce a subsequent generation of pestresistant progeny. In one embodiment, multiple rounds of selfpollination or selfing may be carried out to generate 2, 3, 4, 5 or moregenerations of progeny. The resultant progeny may be tested for pestresistance to ensure that expression of the resistance trait is stablymaintained and inherited.

In a further embodiment, any pest resistant progeny plants derived froma cross between a first transgenic parent plant carrying a DNA constructof interest and a second parent plant lacking said DNA construct may beback-crossed to the second parent plant and subsequent progeny testedfor resistance to pest infestation. Plants or their progeny may betested for resistance to pest infestation either by phenotypic analysisas described elsewhere herein or by standard molecular techniques. Forexample, the pest resistant plants may be identified by the detection ofthe presence of a DNA construct comprising a polynucleotide sequenceencoding an interfering RNA that functions upon uptake by an insect pestspecies to down-regulate expression of a target gene. Techniques fordetecting the presence of specific polynucleotide sequences within cellsare known in the art and include PCR, enzymatic digestion and SNPanalysis.

The methods of the invention can be used to generate ‘stackedtransgenic’ plants that are resistant to insect pest species and thatoptionally possess at least one other desirable trait. As used herein, a‘stacked’ transgenic plant refers to a plant carrying more than oneexogenous polynucleotide sequence. The phrase ‘more than one’ refers tothe possibility of a plant carrying at least 2, at least 3, at least 4exogenous polynucleotides. In one embodiment, the plant cell transformedwith the DNA construct encoding the interfering RNA targeting a pestgene may have previously been engineered to carry a separate exogenouspolynucleotide. Alternatively, the method for generating a transgenicplant from a plant cell as described herein may comprise aco-transformation protocol wherein the DNA construct encoding aninterfering RNA of the invention is delivered to a plant cellsimultaneously or sequentially with a separate exogenous polynucleotide.

Stacked transgenic plants demonstrating pest resistance may also begenerated by standard plant breeding techniques. In one embodiment, afirst pest-resistant transgenic plant is crossed with a second plantengineered to express an exogenous polynucleotide or heterologous geneconferring a desirable plant trait. Any progeny produced can be testedfor pest resistance and acquisition of the additional desirable trait.Alternatively or in addition, any pest-resistant progeny produced fromthe cross may be back-crossed to the second parent in order to generatefurther progeny that can be selected for inheritance of the heterologousgene carried by the second parent and thus the additional desirableplant trait. The exogenous polynucleotides carried by a stackedtransgenic plant of the invention may be expressed in the same parts ofthe plant or may be expressed differentially by virtue of the fact thatexpression of each is controlled by a different tissue-specificpromoter.

In one embodiment, the exogenous polynucleotide or heterologous geneconferring a further desirable trait encodes another interfering RNAtargeting the same or different insect pest species. In a furtherembodiment, the heterologous gene encodes a protein harmful or toxic toa plant insect pest species, for example an insecticidal proteinselected from the group including but not limited to Cry1Ab, Cry1C,Cry2Aa, Cry3, CryET70, Cry22, CryET33, CryET34, CryET80, CryET76,TIC100, TIC101, TIC851, TIC900, TIC901, TIC1201, TIC407, TIC417, PS149B1and VIP insecticidal proteins. In a yet further embodiment, theheterologous gene encodes a protein conferring herbicide resistance.Examples of genes conferring herbicide resistance include Bar, EPSPSwhich confers glyphosate resistance, ALS which confers imidazolinone andsulphonylurea resistance and bxn which confers bromoxynil resistance.

Also provided herein is a method for producing hybrid seed from any ofthe transgenic plants generated by the methods of the current invention,said method comprising the steps of (i) planting the seed obtained froma first inbred plant and the seed obtained from a second inbred plant,wherein at least one of the inbred plants is a transgenic plantresistant to pest infestation (ii) cultivating the seeds into plantsthat bear flowers, (iii) preventing self-pollination of at least one ofthe first or second adult plants, (iv) allowing cross-pollination tooccur between the first and second plants; and (v) harvesting the seedsresulting from the cross-pollination. Hybrid seed produced by thismethod and hybrid plants produced by cultivating said seed are withinthe scope of the current invention. Hybrid plants produced by thismethod will typically be genetically uniform and are likely to exhibitheterosis or hybrid vigour. Thus, crops with the potential for increasedyield may be generated by such a method.

According to another aspect of the current invention are providedtransgenic plants resistant to infestation by insect pest species. Inparticular, provided herein are transgenic plants which express or arecapable of expressing at least one interfering ribonucleic acid (RNA)that functions upon uptake by an insect pest species to down-regulatethe expression of a target gene as described elsewhere herein withinsaid pest. The interfering RNA may be any of those as disclosedelsewhere herein. Preferably, the interfering RNA comprises or consistsof at least one silencing element and said silencing element is a regionof double-stranded RNA comprises annealed complementary strands, onestrand of which (the sense strand) comprises a sequence of nucleotideswhich is at least partially complementary to a target nucleotidesequence within a target gene. Down-regulation of a pest target gene canbe used to disrupt an essential biological process or function in thepest, wherein ‘essential’ refers to the fact that the process orfunction is required for initiation or maintenance of pest infestation.

As used herein, the term ‘plant’ may include any reproductive orpropagation material for a plant. Reference to a plant may also includeplant cells, plant protoplasts, plant tissue cultures, plant calli,plant clumps and plant cells that are intact in plants or parts ofplants such as embryos, pollen, ovules, seeds, leaves, flowers,branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tipsand the like. Progeny, variants and mutants of any of the transgenicplants described herein are within the scope of the current invention.Also included is seed produced from any of said transgenic plants.

Included within the group of transgenic plants of the current inventionare transgenic plants produced by any of the methods described herein.Thus in one embodiment of the invention the transgenic plants comprisestacked transgenic traits carrying a first exogenous polynucleotideconferring pest resistance and at least one other exogenouspolynucleotide or heterologous gene conferring an additional desirableplant trait. The additional heterologous genes may comprise genesencoding additional pesticidal agents, genes encoding proteins toxic orharmful to insect pest species and/or genes encoding proteins conferringherbicide resistance as described elsewhere herein.

Also provided herein is the use of the interfering ribonucleic acid(RNA) as described herein or the DNA construct as described herein forpreventing and/or controlling insect pest infestation, preferably insectpest infestation of plants.

The invention will be further understood with reference to the followingnon-limiting examples.

EXAMPLES Example 1 Identification of Target Genes in Insect Pest Species

1.1 Cloning of Partial Sequences of the Leptinotarsa decemlineata Genes

Nucleic acids were isolated from the gut cells of Colorado potatobeetle, CPB, (Leptinotarsa decemlineata) larvae and a cDNA library wasprepared. The gut cDNAs were cloned into the pGN49A vector (as describedin WO01/88121) such that they were flanked by chemically-inducible T7promoters, oppositely oriented at each end of the cDNA duplex. Therecombinant vector constructs were transformed into cells of Escherichiacoli strain AB301-105 (DE3). The transformed cells were subsequentlydiluted and plated so as to obtain single colonies or clones. The cloneswere checked to ensure that clone redundancy for the library did notexceed 5%. Between 3000 and 4000 clones were generated.

1.2 Testing the Effect of Bacterially-Expressed dsRNA Molecules on theSurvival of Leptinotarsa decemlineata Larvae

Multiwell plates were pre-filled with LB (Luria-broth) medium and eachwell was inoculated with a separate clone of transformed bacterial cellstaken from the CPB cDNA library. The bacteria were grown at 28° C. withshaking at 280 rpm, followed by a chemical induction step at 37° C.After centrifugation, the resulting bacterial pellet was washed withwater and subjected to a heat treatment to inactivate the bacteria.

The bacterial suspensions were tested for their effects on CPB larvaesurvival using an artificial diet-based feeding assay. A bacterialsuspension produced from a single bacterial clone was topically appliedto solid artificial diet in the wells of a multiwell plate and the dietwas dried in a laminar flow cabinet. The effects of each clone weretested in triplicate. A single 2nd stage CPB larva was subsequentlyadded to each well. The plates were stored in an insect rearing chamberat 25±2° C., 60±5% relative humidity, with a 16:8 hours light:darkphotoperiod. Bacterial cells transformed with an empty vector, pGN29(WO01/88121), were used as controls. The survival of CPB larvae in eachof the wells was assessed on day 14.

A number of bacterial clones exhibited high potency against CPB larvaeindicating that the cDNAs encoding double-stranded RNAs containedtherein are essential for pest survival and thus represent target genesof interest for the purposes of pest control. The DNA sequences andcorresponding amino acid sequences of these target genes were thereforedetermined and are provided in Tables 1 and 2 respectively.

TABLE 1 cDNA Sequence (sense strand) Target ID 5′→3′ Ld556 SEQ ID NO 1Ld513 SEQ ID NO 2 Ld504.2 SEQ ID NO 3 Ld520 SEQ ID NO 4 Ld537 SEQ ID NO5 Ld334 SEQ ID NO 6 Ld327 SEQ ID NO 7 Ld502 SEQ ID NO 8 Ld516 SEQ ID NO9 Ld579 SEQ ID NO 10 Ld332 SEQ ID NO 11 Ld237 SEQ ID NO 12 Ld261 SEQ IDNO 13 Ld300.1 SEQ ID NO 14 Ld423 SEQ ID NO 15 Ld511 SEQ ID NO 16 Ld512SEQ ID NO 17 Ld563 SEQ ID NO 18 Ld105 SEQ ID NO 19 Ld248 SEQ ID NO 20

TABLE 2 Corresponding amino acid sequence of Target ID cDNA clone asrepresented in Table 1 Ld556 SEQ ID NO 206 (frame +2) Ld513 SEQ ID NO207 (frame +3) Ld504.2 SEQ ID NO 208 (frame +2) Ld520 SEQ ID NO 209(frame +3) Ld537 SEQ ID NO 210 (frame +1) Ld334 SEQ ID NO 211 (frame +1)Ld327 SEQ ID NO 212 (frame +3) Ld502 SEQ ID NO 213 (frame +2) Ld516 SEQID NO 214 (frame +3) Ld579 SEQ ID NO 215 (frame +1) Ld332 SEQ ID NO 216(frame +3) Ld237 SEQ ID NO 217 (frame +2) Ld261 SEQ ID NO 218 (frame +3)Ld300.1 SEQ ID NO 219 (frame +3) Ld423 SEQ ID NO 220 (frame +1) Ld511SEQ ID NO 221 (frame +3) Ld512 SEQ ID NO 222 (frame +3) Ld563 SEQ ID NO223 (frame +1) Ld105 SEQ ID NO 224 (frame +1) Ld248 SEQ ID NO 225 (frame+1)

1.3 Design of Degenerate Primers for Cloning Lygus hesperus OrthologousSequences

A protein (blastp) search was performed for each target gene of interestby searching non-redundant protein databases of Arthropods using theprotein sequence corresponding to each of the Leptinotarsa decemlineatatargets. A selection of up to twenty protein sequences was made from thebest hits which represented a diversity of insect species.

These sequences were first processed into blocks using the “Blockmaker”program (http://bioinfo.weizmann.ac.il/blockmkr-bin/makeblocks.pl) whichgenerated multiple sequence alignments and analyses of the proteinsequences for regions of conservation.

Blocks of conserved amino acid sequences were submitted to CodeHop(http://blocks.fhcrc.org/codehop.html). From the output of degenerateprimers a selection of up to ten forward and ten reverse primers foreach target was produced.

1.4 Cloning of Partial Sequences of the Lygus hesperus Genes by FamilyPCR

High quality, intact RNA was isolated from 1st instar nymphs of Lygushesperus. Any genomic DNA present in the RNA preparation was removed byDNase treatment. cDNA was generated using Reverse Transcriptase. Toisolate cDNA sequences comprising a segment of the Lh513, Lh504.2,Lh520, Lh537, Lh334, Lh327, Lh579, Lh332, Lh237, Lh261, Lh300.1, Lh423,Lh512, Lh105 and Lh248 genes, a series of PCR reactions with degenerateprimers was performed.

The resulting PCR fragments were analyzed on agarose gel, purified andsequenced. The sequences of the resulting PCR products are representedby the respective SEQ ID NOs as provided in Table 3 and are referred toas the partial sequences. The corresponding partial amino acid sequencesare represented by the respective SEQ ID NOs as provided in Table 4.

TABLE 3 cDNA Sequence (sense strand) Target ID 5′→3′ Lh513.1 SEQ ID NO121 Lh513.2 SEQ ID NO 122 Lh504.2 SEQ ID NO 123 Lh520 SEQ ID NO 124Lh537.1 SEQ ID NO 125 Lh537.2 SEQ ID NO 126 Lh537.3 SEQ ID NO 127Lh537.4 SEQ ID NO 128 Lh537.5 SEQ ID NO 129 Lh334 SEQ ID NO 130 Lh327SEQ ID NO 131 Lh579.1 SEQ ID NO 132 Lh579.2 SEQ ID NO 133 Lh332 SEQ IDNO 134 Lh237 SEQ ID NO 135 Lh261 SEQ ID NO 136 Lh300.1 SEQ ID NO 137Lh423 SEQ ID NO 138 Lh423.2 SEQ ID NO 253 Lh512.1 SEQ ID NO 139 Lh512.2SEQ ID NO 140 Lh105 SEQ ID NO 251 Lh248 SEQ ID NO 252

TABLE 4 Corresponding amino acid sequence of cDNA clone as Target IDrepresented in Table 3 Lh513.1 SEQ ID NO 226 Lh513.2 SEQ ID NO 227Lh504.2 SEQ ID NO 228 Lh520 SEQ ID NO 229 Lh537.1 SEQ ID NO 230 Lh537.2and SEQ ID NO 231 Lh537.3 Lh537.4 SEQ ID NO 232 Lh537.5 SEQ ID NO 233Lh334 SEQ ID NO 234 Lh327 SEQ ID NO 235 Lh579.1 SEQ ID NO 236 Lh579.2SEQ ID NO 237 Lh332 SEQ ID NO 238 Lh237 SEQ ID NO 239 Lh261 SEQ ID NO240 Lh300.1 SEQ ID NO 241 Lh423 SEQ ID NO 242 Lh423.2 SEQ ID NO 271Lh512.1 SEQ ID NO 243 Lh512.2 SEQ ID NO 244 Lh105 SEQ ID NO 269 Lh248SEQ ID NO 270

1.5 Full Length cDNA Cloning by RACE (Rapid Amplification of cDNA Ends)

In order to clone full length cDNA, starting from a known clone ofinternal fragment from the most potent targets, the 5′/3′ RACE kit wasused (Roche, Cat. No. 1 734 792; based on Sambrook, J. & Russell, D. M).The standard protocol, described in the Instruction Manual, wasfollowed. Briefly, for a 5′ RACE, a target sequence specific antisenseprimer was designed on the known sequence and used for a first strandcDNA synthesis, using Lygus RNA as template. A tail was added to thefirst strand cDNA and used as an anchor for the second strand synthesisand amplification of an unknown end portion of the transcript. For a 3′RACE, an oligo dT anchor primer was used for the first strand cDNAsynthesis. For the 5′ and 3′ RACEs, nested primers, specific to thetarget sequence were used in a second PCR reaction. The PCR fragmentswere analysed on agarose gel, purified, cloned and sequenced forconfirmation.

Full length cDNA sequences corresponding to the Lygus targets listed inTable 13 were assembled in VectorNTi, a fully integrated sequenceanalysis software package for DNA sequence analysis (Invitrogen). Thenucleotide sequences resulting from the assemblies are provided in Table14 and the corresponding amino acid sequences are provided in Table 15.

TABLE 13 Target ID Dm orthologue NAME SYMBOL Lh049 CG8055 shrub - snf7(vesicle trafficking) shrb Lh248 CG6699 beta′-coatomer protein beta′CopLh105 CG1250 5ec23 5ec23 Lh300 CG6213 vacuolar H[+] ATPase G-subunitVha13 Lh327 CG6223 beta-coatomer protein betaCop Lh423 CG2746 ribosomalprotein L19 RpL19 Lh504 CG5271 ribosomal protein 527A RpS27A Lh520CG2960 ribosomal protein L40 RpL40

TABLE 14 cDNA Sequence Target ID (sense strand) 5′→3' Lh049 SEQ ID NO280 Lh248 SEQ ID NO 279 Lh105 SEQ ID NO 278 Lh300 SEQ ID NO 276 Lh327SEQ ID NO 275 Lh423 SEQ ID NO 277 Lh504 SEQ ID NO 273 Lh520 SEQ ID NO274

TABLE 15 Corresponding amino acid sequence to cDNA, as Target IDrespresented in Table 14 Lh049 SEQ ID NO 288 Lh248 SEQ ID NO 287 Lh105SEQ ID NO 286 Lh300 SEQ ID NO 284 Lh327 SEQ ID NO 283 Lh423 SEQ ID NO285 Lh504 SEQ ID NO 281 Lh520 SEQ ID NO 282

Example 2 In Vitro Production of Double-Stranded RNAs for Gene Silencing

2.1 Production of dsRNAs Corresponding to the Partial Sequences of theLeptinotarsa decemlineata and Lygus hesperus Target Genes

Double-stranded RNA was synthesized in milligram quantities. First, twoseparate 5′ T7 RNA polymerase promoter templates (a sense template andan antisense template) were generated by PCR. PCRs were designed andcarried out so as to produce sense and antisense templatepolynucleotides, each having the T7 promoter in a different orientationrelative to the target sequence to be transcribed.

For each of the target genes, the sense template was generated using atarget-specific T7 forward primer and a target-specific reverse primer.The antisense templates were generated using target-specific forwardprimers and target-specific T7 reverse primers. The sequences of therespective primers for amplifying the sense and antisense templates viaPCR for each of the target genes are provided in Table 5 (forLeptinotarsa decemlineata cDNAs) and in Table 6 (for Lygus hesperuscDNAs). The PCR products were analysed by agarose gel electrophoresisand purified. The resultant T7 sense and antisense templates were mixedand transcribed by the addition of T7 RNA polymerase. Thesingle-stranded RNAs produced by transcription from the templates wereallowed to anneal, were treated with DNase and RNase, and were purifiedby precipitation. The sense strand of the resulting dsRNA produced fromeach of the target genes is provided in Table 5 (for Leptinotarsadecemlineata) and in Table 6 (for Lygus hesperus).

TABLE 5 dsRNA: sense strand represented Primers Primers by equivalentForward Reverse DNA Sequence Target ID 5′→3′ 5′→3′ 5′→3′ Ld556 SEQ ID NO41 SEQ ID NO 42 SEQ ID NO 21 SEQ ID NO 43 SEQ ID NO 44 Ld513 SEQ ID NO45 SEQ ID NO 46 SEQ ID NO 22 SEQ ID NO 47 SEQ ID NO 48 Ld504.2 SEQ ID NO49 SEQ ID NO 50 SEQ ID NO 23 SEQ ID NO 51 SEQ ID NO 52 Ld520 SEQ ID NO53 SEQ ID NO 54 SEQ ID NO 24 SEQ ID NO 55 SEQ ID NO 56 Ld537 SEQ ID NO57 SEQ ID NO 58 SEQ ID NO 25 SEQ ID NO 59 SEQ ID NO 60 Ld334 SEQ ID NO61 SEQ ID NO 62 SEQ ID NO 26 SEQ ID NO 63 SEQ ID NO 64 Ld327 SEQ ID NO65 SEQ ID NO 66 SEQ ID NO 27 SEQ ID NO 67 SEQ ID NO 68 Ld502 SEQ ID NO69 SEQ ID NO 70 SEQ ID NO 28 SEQ ID NO 71 SEQ ID NO 72 Ld516 SEQ ID NO73 SEQ ID NO 74 SEQ ID NO 29 SEQ ID NO 75 SEQ ID NO 76 Ld579 SEQ ID NO77 SEQ ID NO 78 SEQ ID NO 30 SEQ ID NO 79 SEQ ID NO 80 Ld332 SEQ ID NO81 SEQ ID NO 82 SEQ ID NO 31 SEQ ID NO 83 SEQ ID NO 84 Ld237 SEQ ID NO85 SEQ ID NO 86 SEQ ID NO 32 SEQ ID NO 87 SEQ ID NO 88 Ld261 SEQ ID NO89 SEQ ID NO 90 SEQ ID NO 33 SEQ ID NO 91 SEQ ID NO 92 Ld300.1 SEQ ID NO93 SEQ ID NO 94 SEQ ID NO 34 SEQ ID NO 95 SEQ ID NO 96 Ld423 SEQ ID NO97 SEQ ID NO 98 SEQ ID NO 35 SEQ ID NO 99 SEQ ID NO 100 Ld511 SEQ ID NO101 SEQ ID NO 102 SEQ ID NO 36 SEQ ID NO 103 SEQ ID NO 104 Ld512 SEQ IDNO 105 SEQ ID NO 106 SEQ ID NO 37 SEQ ID NO 107 SEQ ID NO 108 Ld563 SEQID NO 109 SEQ ID NO 110 SEQ ID NO 38 SEQ ID NO 111 SEQ ID NO 112 Ld105SEQ ID NO 113 SEQ ID NO 114 SEQ ID NO 39 SEQ ID NO 115 SEQ ID NO 116Ld248 SEQ ID NO 117 SEQ ID NO 118 SEQ ID NO 40 SEQ ID NO 119 SEQ ID NO120

TABLE 6 dsRNA: sense strand represented by equivalent Target PrimersForward Primers Reverse DNA Sequence ID 5′→3′ 5′→3′ 5′→3′ Lh513 SEQ IDNO 154 SEQ ID NO 155 SEQ ID NO 141 SEQ ID NO 156 SEQ ID NO 157 Lh504.2SEQ ID NO 158 SEQ ID NO 159 SEQ ID NO 142 SEQ ID NO 160 SEQ ID NO 161Lh520 SEQ ID NO 162 SEQ ID NO 163 SEQ ID NO 143 SEQ ID NO 164 SEQ ID NO165 Lh537 SEQ ID NO 166 SEQ ID NO 167 SEQ ID NO 144 SEQ ID NO 168 SEQ IDNO 169 Lh334 SEQ ID NO 170 SEQ ID NO 171 SEQ ID NO 145 SEQ ID NO 172 SEQID NO 173 Lh327 SEQ ID NO 174 SEQ ID NO 175 SEQ ID NO 146 SEQ ID NO 176SEQ ID NO 177 Lh579 SEQ ID NO 178 SEQ ID NO 179 SEQ ID NO 147 SEQ ID NO180 SEQ ID NO 181 Lh332 SEQ ID NO 182 SEQ ID NO 183 SEQ ID NO 148 SEQ IDNO 184 SEQ ID NO 185 Lh237 SEQ ID NO 186 SEQ ID NO 187 SEQ ID NO 149 SEQID NO 188 SEQ ID NO 189 Lh261 SEQ ID NO 190 SEQ ID NO 191 SEQ ID NO 150SEQ ID NO 192 SEQ ID NO 193 Lh300.1 SEQ ID NO 194 SEQ ID NO 195 SEQ IDNO 151 SEQ ID NO 196 SEQ ID NO 197 Lh423 SEQ ID NO 198 SEQ ID NO 199 SEQID NO 152 SEQ ID NO 200 SEQ ID NO 201 Lh512 SEQ ID NO 202 SEQ ID NO 203SEQ ID NO 153 SEQ ID NO 204 SEQ ID NO 205 Lh105.2 SEQ ID NO 257 SEQ IDNO 258 SEQ ID NO 254 SEQ ID NO 259 SEQ ID NO 260 Lh248.2 SEQ ID NO 261SEQ ID NO 262 SEQ ID NO 255 SEQ ID NO 263 SEQ ID NO 264 Lh248.3 SEQ IDNO 265 SEQ ID NO 266 SEQ ID NO 256 SEQ ID NO 267 SEQ ID NO 268 GFP SEQID NO 246 SEQ ID NO 247 SEQ ID NO 245 SEQ ID NO 248 SEQ ID NO 249

Example 3 Down-Regulating Expression of Target Genes in Leptinotarsadecemlineata as a Means to Achieve Pest Control

3.1 Testing of In Vitro-Synthesized dsRNA Molecules for Activity AgainstLeptinotarsa decemlineata Larvae

In vitro-synthesized dsRNAs transcribed from template polynucleotidesderived from target genes identified according to the methods of Example1 were tested for effects on the survival of CPB larvae using feedingassays. Briefly, artificial diet-based assays were carried out asfollow. To each well of a 48-well plate was added 0.5 ml artificial dietpre-treated by topical application of 1000 ng/cm² synthetic dsRNA in 25μL. A single L2 larva was added to each well. dsRNAs derived from avariety of target genes were tested using this method and 24 larvae weretested for each dsRNA. The total numbers of surviving larvae werecounted at regular intervals up to 14 days after the start of feeding.The time taken for each dsRNA to kill 50% of treated larvae (LT₅₀) wascalculated for each target gene investigated and compared to the LT₅₀for a reference target gene (Ld248) previously described inWO2007/074405. The results are presented in Table 7.

TABLE 7 LT₅₀ target X/ dsRNA Dm¹ LT₅₀ target length Rank Targetorthologue Name / Description (FlyBase) Ld248 (bp) 1 Ld556 CG11415Tsp2A; Tetraspanin 2A, putative 0.60 558 transmembrane domain protein 2Ld513 CG5409 & Protein belonging to the actin 0.67 320 others family;structural constituent of cytoskeleton 3 Ld504.2 CG5271 Ribosomalprotein S27A; 0.70 490 structural constituent of ribosome 4 Ld520 CG2960Ribosomal protein L40; structural 0.70 231 constituent of ribosome 5Ld537 CG32744 Ubiquitin-5E; protein modification 0.77 571 process 6Ld334 CG3948 ζ-coatomer of COPI vesicle 0.80 542 7 Ld327 CG6223β-coatomer of COPI vesicle 0.83 747 8 Ld105 CG1250 Sec23; GTPaseactivator involved 0.85 1504 in intracellular protein transport 9 Ld502CG7595 Crinkled protein; unconventional 0.90 393 myosin involved inmotor activity 10 Ld516 CG11888 Rpn2; proteasome regulatory 0.90 779particle 11 Ld579 CG8392 Proteasome β1 subunit 0.90 392 12 Ld332 CG1528y-coatomer of COPI vesicle 0.90 178 13 Ld237 CG10149 Rpn6; proteasomep44.5 subunit 0.90 559 14 Ld261 CG5266 Pros25; proteasome 25kD subunit0.90 586 15 Ld300.1 CG6213 Vacuolar H⁺-ATPase G-subunit 1.00 267 16Ld248 CG6699 β′-coatomer protein 1.00 967 17 Ld423 CG2746 Ribosomalprotein L19 1.00 603 18 Ld511 CG3329 Prosβ2; proteasome β2 subunit 1.02273 ¹Drosophila melanogaster

From these results, it can be concluded that dsRNAs targeting the CPBgenes identified in Example 1 are more potent or at least as potent asreference CPB target genes that have been previously reported inWO2007/074405 i.e. in the majority of cases, the ‘time-to-kill’ CPBlarvae was shorter than for the reference targets Ld105 and Ld248 (aspreviously described in WO2007/074405: Ld105=[Target ID NO LD010],Ld248=[Target ID NO LD027]). The efficacy against CPB larvae after dsRNAconsumption differed depending on the nature of the gene targeted.

3.2 Testing of In Vitro-Synthesized dsRNA Molecules for Activity AgainstLeptinotarsa decemlineata Adults

The data provided below exemplify the findings that ingested dsRNAsproduced from template polynucleotides derived from a variety of CPBtarget genes adversely affected the survival, fitness, feedingbehaviour, mating behaviour, egg production and offspring generation ofCPB adults.

Various gene targets selected from the larvae gut cDNA expressionlibrary and as shown above in Example 3.1 to be effective for thepurposes of killing CPB larvae, were tested in order to assess theeffects of gene down-regulation in adult beetles. Gene targetsidentified as important in both CPB larvae and adult beetles are ofparticular interest since they may be used to control and/or preventinsect pest infestation at different stages of an insect's life-cycle.

Briefly, a leaf disc assay to test the effects of dsRNA moleculesderived from various target genes, against adult beetles was set up asfollows. A potato leaf disc, diameter 1.5 cm, treated with 5 μg of invitro-synthesized target dsRNA provided in 20 μL, was placed in a wellof a 6-well plate together with a week-old CPB adult. After a few hours,once the leaf disc had been entirely consumed by the insect, anotherleaf disc treated with 5 μg of in vitro-synthesized target dsRNA waspresented to the adult. For each dsRNA to be tested, a total of tenyoung adult beetles (a mix of males and females) were exposed to treatedleaf discs. The following day, after the second treated leaf disc hadbeen entirely consumed, the ten adults in each treatment group werepooled in the same box and fed abundant untreated potato foliage. Adultbeetles fed dsRNA derived from the GFP gene were used as controls inthis assay.

The numbers of surviving and/or moribund adult insects were counted atregular intervals from day 4 up to 14 days after the start of thefeeding assay. CPB adults were classified as moribund if they appearedsick and less mobile than healthy adults and were not able to rightthemselves when placed on their backs for longer than one minute.Effects on feeding, mating, egg production and hatching were alsoassessed at regular intervals from day 4.

Mortality and Moribundity

The percentage of moribund and/or dead CPB adults assessed over a 14-dayperiod for each target gene tested are shown in FIG. 1. In addition, thetime taken to achieve 50% mortality of adult beetles (LT₅₀) in eachtreatment group was calculated and the results shown in Table 8 arepresented as a ratio based on the LT₅₀ calculated for a reference targetgene previously described in WO2007/074405.

TABLE 8 LT50 target X/ dsRNA Dm LT50 target length Rank Targetorthologue Name / Description (FlyBase) Ld248 (bp) 1 Ld537 CG32744Ubiquitin-5E; protein modification 0.63 571 process 2 Ld520 CG2960Ribosomal protein L40; structural 0.70 231 constituent of ribosome 3Ld516 CG11888 Rpn2; proteasome regulatory 0.74 779 particle 4 Ld512CG10370 Tbp-1; Tat-binding protein 0.74 310 5 Ld511 CG3329 Prosβ2;proteasome β2 subunit 0.77 273 6 Ld579 CG8392 Proteasome β1 subunit 0.87392 7 Ld513 CG5409 & Protein belonging to the actin family; 0.88 320others structural constituent of cytoskeleton 8 Ld248 CG6699 β-coatomerprotein 1.00 967 9 Ld563 CG3193 Crooked neck protein; involved in 1.05388 regulation of nuclear alternative mRNA splicing 10 Ld105 CG1250Sec23; GTPase activator involved in 1.06 1504 intracellular proteintransport

From these results, it can be concluded that dsRNAs targeting a varietyof CPB genes identified in Example 1 are more potent or at least aspotent as reference CPB target genes that have been previously reportedin the WO2007/074405 i.e. in the majority of cases, the ‘time-to-kill’CPB adults was shorter than for the reference targets Ld105 and Ld248(as previously described in WO2007/074405: Ld105=[Target ID NO LD010],Ld248=[Target ID NO LD027]). The efficacy against CPB adults after dsRNAconsumption differed depending on the nature of the gene targeted.

Feeding Inhibition

For all target genes of interest, complete cessation of feeding by CPBadults was observed from day 5 onwards of the assay (4 days after thetreated potato foliage was replaced by untreated potato foliage).Control CPB adults fed as normal throughout the assay.

Mating, Egg Production, and Egg Hatching

Mating, egg production, and egg hatching were assessed throughout the14-day time-course of the assay. The results are shown in Table 9.

TABLE 9 Target Day 4 Day 5 Day 6 Day 7 Day 8 Day 14 Ld105 EM SE nonenone none none Ld248 EM YS YS none none none Ld516 EM SE none none nonenone Ld511 EM & SE YS none none none none Ld512 EM & SE SE SE none nonenone Ld513 EM & SE none none none none none Ld520 SE none none none nonenone Ld537 EM & SE SE none none none none Ld563 none SE none none nonenone Ld579 EM SE SE none none none GFP EM EM EM EM EM EM

From day 5 onwards, no mating was observed in CPB adults in the treatedgroups, however, mating of CPB adults in the GFP control group was asnormal.

On day 4, normal egg masses (with 20-30 eggs per egg mass) were observedin groups treated with dsRNAs targeting Ld105, Ld248, Ld516, Ld579, andGFP. (These egg masses were laid between days 1 and 3 of the assay.)However, for the groups treated with dsRNAs targeting Ld511, Ld512,Ld513, and Ld537, a mixture of normal egg masses and single eggs werefound. No normal egg masses were observed in the CPB group treated withdsRNA targeting Ld520; only individual single eggs were observed.

Eggs identified on day 4 were harvested and both hatching frequency andlarval survival were assessed. In all treatment groups for which normalegg masses were harvested, hatching was 100% except for the Ld537 groupin which only 50% of eggs in the single egg mass harvested went on tohatch. All larvae from these egg masses developed normally.

For groups in which Ld511 and Ld537 were the target genes of interest,less than 10% of the single eggs hatched and these larvae died within 7days after hatching. For the groups in which the target genes wereLd512, Ld520 and Ld513, the hatching frequency was 30%, 70% and 100%respectively.

On day 5, no egg masses were observed in any of the treatment groupsexcept for the GFP control. However, single eggs were present in thegroups wherein the target genes were Ld105, Ld516, Ld512, Ld537, Ld563,and Ld579. A yellow smear (indicative of no intact eggs) was visible inthe feeding chambers of insects in the groups treated with dsRNAstargeting Ld248 and Ld511. In the groups for which Ld513 and Ld520 werethe target genes, no eggs were discovered.

Eggs identified on day 5 were harvested and both hatching frequency andlarval survival were assessed. For groups with target genes Ld512,Ld516, and Ld563, hatching was between 20 and 50%, but all larvae ingroup Ld563 died within 7 days of hatching. For the group with Ld579 asthe gene target, all eggs hatched and larvae developed as normal.

On day 6, only single eggs were identified in the treatment groupswherein the target genes were Ld512 and Ld579. A yellow smear was notedin the Ld248 target group. No eggs were present in any of the othertreatment groups. From day 7 onwards, no eggs were laid by any of thetreated adult beetles. However, GFP dsRNA-treated control femalescontinued to lay normal egg masses throughout the assay.

Example 4 Down-Regulating Expression of Target Genes in Lygus hesperusby Ingestion of dsRNA as a Means to Achieve Pest Control

4.1 Modification of the Lygus hesperus Feeding Assay to Allow for MoreEffective Uptake of dsRNA Molecules

Although the rearing diet from BioServ used in maintaining the Lyguscultures over many generations is effective, this diet may not beappropriate for testing candidate dsRNA molecules in a feeding assay.The oligidic diet includes ingredients which are not chemically definedand therefore some components may possibly interact with the moleculesto be delivered to the nymphs. Therefore, the Lygus feeding assay hasbeen modified such that it consists of two phases: an initial exposureof early instar nymphs to dsRNA molecules in a simplified diet (15%sucrose only) for three days followed by transfer of the nymphs to anormal oligidic Lygus diet for the remainder of the assay.

4.2 Effects of Various Target dsRNAs Plus Yeast tRNA on Lygus hesperusSurvival

Lygus hesperus nymphs were exposed to 0.5 μg/μL dsRNA derived a numberof Lygus hesperus target genes as described herein in the presence of 5μg/μL yeast tRNA (Sigma) in a feeding assay (FIG. 2). Controls are GFPdsRNA plus yeast tRNA at the same concentrations, respectively, and dietonly treatments. Young nymphs were each exposed to 25 μL of 15% sucrosediet with or without incorporated test components for three days priorto transferring them on to 50 μL complex (Bioserv) diet. Complex dietwas refreshed on day 7.

In this assay, ingested dsRNA from all tested targets in combinationwith tRNA led to high mortalities of L. hesperus nymphs when compared tothe GFP dsRNA or diet only control treatments (Table 10).

TABLE 10 Log-rank test (versus GFP) DsRNA Significant Target ID length(bp) Chi square P - value difference? Lh520 231 18.04 <0.0001 *** Lh423511 17.11 <0.0001 *** Lh537 300 14.63 0.0001 *** Lh504.2 168 12.990.0003 *** Lh512 495 11.86 0.0006 *** Lh334 172 10.39 0.0013 ** Lh300.1235 10.22 0.0014 ** Lh327 408 9.153 0.0025 ** Lh332 1041 7.972 0.0047 **Lh237 710 5.793 0.0161 * Lh579 273 5.336 0.0209 * Lh261 368 3.9280.0475 * Lh513 625 2.144 0.1432 ns diet / 1.483 0.2233 ns only tsurvival curves significantly different? *** = extremely significant, **= very significant, * = significant, ns = not significant

A table which ranks the targets according to potency is made based uponthe results of this RNAi-by-feeding assay (Table 11 and FIG. 3).

TABLE 11 dsRNA D. melanogaster % Survival Target length (bp) orthologueDescription (FlyBase) range at day 10 Figure Lh423 511 CG2746 Ribosomalprotein L19  0 - 15 3 A Lh520 231 CG2960 Ribosomal protein L40 Lh504.2168 CG5271 Ribosomal protein 16 - 30 3 B S27A Lh537 300 CG32744Ubiquitin-5E Lh512 495 CG10370 Tbp-1, Tat-binding protein Lh300.1 235CG6213 Vacuolar H⁺-ATPase G 3 C subunit Lh327 408 CG6223 β-coatomer ofCOPI vesicle Lh334 172 CG3948 ζ-coatomer of COPI vesicle Lh332 1041CG1528 γ-coatomer of COPI vesicle Lh579 273 CG8392 Proteasome β1 subunit31 - 45 3 D Lh237 710 CG10149 Rpn6, proteasome p44.5 subunit Lh261 368CG5266 Pros25, proteasome >45 3 D 25 kD subunit Lh513 625 CG4027 Actin50

In another RNAi-by-feeding assay under the same conditions as describedhereabove, we tested the effects of Lh105 and Lh248 target dsRNA onLygus hesperus nymphal survival. Double-stranded RNA from targets Lh105and Lh248 at 0.5 μg/μL in the presence of 5 μg/μL of tRNA led tosignificant L. hesperus nymphal lethality at day 10 in a feeding assay(83% for Lh105.2 dsRNA and 58% for Lh248.2 dsRNA and 71% for Lh248.3dsRNA) (FIG. 6). In the same assay, Lh327 and Lh300 dsRNA showed only 4%and 13% survivors, respectively, at the end of the bioassay (FIG. 6).

4.3 Dose-Response Relationship Over Time of Target dsRNA Against Lygushesperus Nymphs in RNAi-by-Feeding Assays

The dose-response relationship was studied to determine the L. hesperusnymphs' susceptibility to lowering dsRNA concentrations of test targetsin RNAi-by-feeding assays. The data are graphically represented in FIGS.4 and 5. The Kaplan-Meier estimated survival curves of the test targetsat the low concentrations of 0.1, 0.05 and 0.025 μg/μL were comparedwith those of GFP dsRNA control at 0.1 μg/μL using the log rank test(Table 12).

TABLE 12 Target dsRNA1 Dose (μg/μL) X² P-value Significance Lh423 0.130.36 <0.0001 *** 0.05 6.759 0.0093 ** 0.025 6.239 0.0125 * Lh504.2 0.15.828 0.0158 * 0.05 0.7283 0.3934 ns 0.025 0.4834 0.4869 ns Lh537 0.12.150 0.1426 ns 0.05 2.150 0.1426 ns 0.025 0.007874 0.9293 ns Lh327 0.113.80 0.0002 *** 0.05 6.176 0.0129 * 0.025 3.000 0.0833 ns Lh520 0.112.65 0.0004 *** 0.05 2.944 0.0862 ns 0.025 1.893 0.1689 ns Lh300.1 0.112.24 0.0005 *** 0.05 10.81 0.0010 ** 0.025 1.615 0.2038 ns 1in thepresence of 5 pg/i..iL of yeast transfer RNA

All targets tested were toxic towards L. hesperus nymphs atconcentrations as low as 0.1 μg/μL. Target Lh423 dsRNA at thisconcentration yielded under 10% nymphal survival towards the end of thebioassays. At the lowest concentration tested, i.e. 0.025 μg/μL, targetLh423 still showed a significant drop in survival when compared to GFP.Double-stranded RNAs from targets Lh300.1 and Lh327 also demonstratedhigh potency at low concentrations with significant drops in survival at0.05 μg/μL.

Example 5 Generation of Plants Resistant to Insect Pest Species

5.1 Assembly of Plant Expression Vectors Comprising a Lygus hesperusHairpin Sequence or Leptinotarsa decemlineata Hairpin Sequence forTransformation of Potato or Cotton

Since the mechanism of RNA interference operates through dsRNAfragments, the target polynucleotide sequences were cloned in anti-senseand sense orientation, separated by an intron (SEQ ID NO 250), to form adsRNA hairpin construct. The dsRNA hairpin constructs encoding the L.hesperus dsRNA molecule derived from the target genes as mentionedherein were subcloned into a plant expression vector. Similarly a GUSdsRNA hairpin control construct, wherein the sense polynucleotidesequence encoding GUS (SEQ ID NO 272) was cloned in anti-sense and senseorientation, separated by the same intron (SEQ ID NO 250), was subclonedinto a plant expression vector.

The plant expression vector comprises as well elements necessary for themaintenance of the plasmid in a bacterial cell. The dsRNA hairpinconstruct is located between the left border (LB) and right border (RB),3′ downstream from the Cauliflower Mosaic Virus 35S promoter (P35S) and5′ upstream from the TNOS terminator. A GFP reporter expression cassettecomprising the GFP sequence flanked by the P35S promoter and terminatorwas subcloned into the plant transformation vector harbouring the L.hesperus hairpin cassette. The NPT II expression cassette comprising theNPT II sequence flanked by the P35S promoter and terminator is used forselecting plants that have been effectively transformed. Correctassembly of the genetic fragments in the plant expression vector wasconfirmed by sequencing (FIG. 7).

The plant expression vectors comprising the individual L. hesperustarget hairpins were subsequently transformed into Agrobacteriumtumefaciens. For all L. hesperus target genes mentioned herein,fragments can be selected and cloned as hairpins in a similar manner.

5.2 Transformation of Potato with a Plant Expression Vector Comprising aLygus hesperus Hairpin Sequence or Leptinotarsa decemlineata HairpinSequence and Testing of the Transformed Potato Plants for ResistanceTowards L. hesperus or Leptinotarsa decemlineata

The example provided below is an exemplification of the finding thattransgenic potato plants expressing target gene-specific hairpin RNAsadversely affect survival and/or development of insect pest species.

Lygus hesperus RNAi-by-Feeding in Planta

Potato Transformation

Stably transformed potato plants were obtained using an adapted protocolreceived through Julie Gilbert at the NSF Potato Genome Project(http://www.potatogenome.org/nsf5). Stem internode explants of potato‘Line V’ (originally obtained from the Laboratory of Plant Breeding atPRI Wageningen, the Netherlands) which was derived from the susceptiblediploid Solanum tuberosum 6487-9 was used as starting material fortransformation. In vitro-derived explants were inoculated withAgrobacterium tumefaciens C58C₁Rif^(R) containing the hairpinconstructs. After three days co-cultivation, the explants were put ontoa selective medium containing 100 mg/L Kanamycin and 300 mg/L Timentin.After 6 weeks post-transformation, the first putative shoots wereremoved and rooted on selective medium. Shoots originating fromdifferent explants were treated as independent events, shootsoriginating from the same callus were termed ‘siblings’ until theirclonal status could be verified by Southern blotting, and nodal cuttingsof a shoot were referred to as ‘clones’.

The transgenic status of the rooting shoots was checked either by GFPfluorescence and by plus/minus PCR for the inserted target sequence.Positive shoots were then clonally propagated in tissue culture toensure enough replicates were available for the Lygus hesperus orLeptinotarsa decemlineata assays. These shoots were kept in tissueculture medium for greater flexibility to test for resistance towardsLygus nymphs/adults or L. decemlineata larvae/adults. The first plantswere available to test fourteen weeks post transformation.

Bioassay

Following the positive results obtained in the dsRNA feeding experimentsproof-of-principle in planta experiments were initiated.

The plantlets were analysed by PCR to confirm the integration of theT-DNA and the presence of the hairpin, before being propagated. Excessexplants were produced with the aim of obtaining at least 30 independentevents for each construct.

The in planta assay for Lygus hesperus was developed with in vitropotato plantlets which sustained insect survival at least 8 days,keeping background mortality low. L. hesperus nymphs survived and fed onwild type potato plantlets. This was supported by the visual damagecaused by insects which was observed on the leaves and buds (FIG. 8).

In the assay, L. hesperus was fed with transgenic potato, expressinghairpin dsRNA targeting the L. hesperus targets identified herein.Plasmids carrying hairpin constructs and a GUS control were generated.

Young transgenic potato plants were kept in tissue culture medium in aplant growth room chamber with the following conditions: 25±1° C., 50±5%relative humidity, 16:8 hour light:dark photoperiod. Per construct, anumber of events, in this case 30 (P006/XX), were generated with asuitable number of clones (more than 20) per event. A number of youngLygus nymphs/adults were placed on the individually caged young (forexample, at the 4-5 unfolded leaf stage) potato plants and left for atleast seven days. The resistance towards Lygus hesperus was assessedregularly over the period of the assay in terms of reduced nymph/adultsurvival, delayed development and stunted growth, and/or decreased plantfeeding damage. FIG. 9 specifically shows L. hesperus nymph survival.The transgenic events were compared to the control GUS hairpintransformed events (P001/XX) and the wild type potatoes (FIG. 9 and FIG.10).

A bioassay for testing transgenic plants, transformed with L.decemlineata specific hairpin constructs, for resistance against L.decemlineata larvae or adults is done in the same way as described forL. hesperus.

5.3 Transformation of Cotton with a Plant Expression Vector Comprising aLygus hesperus Hairpin Sequence and Testing of the Transformed CottonCallus Material or Plants for Resistance Towards L. hesperus

The example provided below is an exemplification of the finding thattransgenic cotton plants or callus expressing target gene-specifichairpin RNAs adversely affect survival and/or development of insect pestspecies.

Cotton Transformation

Coker 312 seed is surface sterilized using first, a 5 minute wash in 70%ethanol and then shaking in a 20% bleach (Chlorox Co. USA, 1% availablechlorine) solution plus 10 drops of the non-ionic detergent, Tween® 20,per litre. The seed is then rinsed 3 times in sterile distilled waterbefore blotting dry on sterile filter papers. The sterile seed isgerminated on Seed Germination (SG) medium for 4-6 days, and at thispoint the hypocotyls are harvested and cut into 0.5 cm lengths ready forAgrobacterium inoculation. The cut sections are placed on sterile filterpapers overlaying a Murashige and Skoog based medium containing nophytohormones. The explants are incubated on a 16:8 hours day:nightcycle at 28° C.+/−2° C. for 3 days prior to inoculation.

For the inoculation, an Agrobacterium tumefaciens liquid LB culture (10ml), strain GV3101 (pMP90) Gent^(R) or strain LBA4404 containing the RNAhairpin target of choice and a hygromycin resistance encoding plantselection cassette, is grown up overnight and 5 ml used to inoculate a100 ml culture the evening prior to the inoculation. The culture is spundown, resuspended and diluted to an OD600 of 0.15 in the bacterialdilution medium.

The hypocotyl segments are inoculated with the Agrobacterium suspensionfor 5 minutes. After this the explants are blotted onto sterile filterpaper to remove the excess bacterial suspension. The explants areincubated in the dark on Cotton Co-cultivation Medium (CCM) for 48hours. The explants are then placed on Selective Callus Induction Medium(SCIM) containing 10 mg/l Hygromycin and 500 mg/l Cefotaxime andincluding the phytohormones 2, 4-dichlorophenoxyacetic acid (0.1 μg/ml)and kinetin (0.1 μg/ml). After 4-6 weeks the first resistant calli areobserved, and these can be removed to fresh SCIM and further amplifiedfor molecular assessment and insect bioassays. Plates are refreshedevery 4-6 weeks to maintain nutrients and antibiotic selection.

Calli that are shown to give a positive result in the insect feedingbioassay are chosen for regeneration and callus is transferred tonon-selective medium for the maturation of the somatic embryos, therecipe is based on the publication of Trolinder and Goodin, 1986. Oncethe embryos have reached 4 mm in length and have differentiatedcotyledons and radicles (2-3 months after transfer to maturationmedium), they can be transferred Elongation Rooting Medium. Thisconsists of sterilized vermiculite in large test tubes soaked with aStewart & Hsu (1977) based liquid medium supplemented with kinetin,giberellic acid both added at the final concentration of 0.1 mg/l. Theembryos are incubated at 28° C. in a 16:8 day/night cycle, and once theyreach the 2-3 leaf stage the plantlets can be hardened off in pots ofvermiculite enclosed in a propagator to maintain humidity. Once theplants are fully hardened (4-6 true leaf stage) they can be potted intoa 50:50 peat:loam mix and grown in a 14:10 light cycle at 30/20° C.day/night.

Bioassay

Lygus nymphs are placed in a Petri dish containing undifferentiatedcotton callus tissue expressing target hairpin RNA. Per construct, anumber of transformed cotton calli are generated and tested in a feedingbioassay for reduced nymph/adult survival and/or delayed development andstunted growth. Transgenic calli not expressing Lygus target hairpin RNAgene fragment serve as a negative control. Furthermore, youngregenerated cotton plants from transgenic calli are grown in soil in aplant growth room chamber with the following conditions: 30/20° C.day/night, 50±5% relative humidity, 14:10 hour light:dark photoperiod.Per construct, a number of events (for example, twenty) are generated. Anumber of young Lygus nymphs/adults are placed on the individually cagedyoung (for example, at the 4-5 unfolded leaf stage) plants and left forat least seven days before assessing resistance towards Lygus hesperusin terms of reduced nymph/adult survival, delayed development andstunted growth, and/or decreased plant feeding damage. Cotton plants nottransformed with the Lygus target hairpin RNA gene fragment serve as anegative control.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the above mentionedassays without departing from the spirit or scope of this assay asgenerically described. Those skilled in the art will recognize, or beable to ascertain using no more than routine experimentation, manyequivalents to the specific examples, and such equivalents are intendedto be encompassed by the present invention. The present example,therefore, is to be considered in all respects as illustrative and notrestrictive.

What is claimed:
 1. An isolated polynucleotide encoding a dsRNA, whereinsaid dsRNA comprises annealed complementary strands, one strand of whichcomprises or consists of a sequence of at least 21 contiguousnucleotides that is 100% identical to a target nucleotide sequencewithin the target gene, wherein said target gene: (i) has a nucleotidesequence comprising at least one of SEQ ID NOs 11, 31, 134, and 148, orthe complement thereof, or a nucleotide sequence that, when the twosequences are optimally aligned and compared, is at least 85% identicalto at least one of SEQ ID NOs 11, 31, 134, and 148, or the complementthereof; or (ii) is an insect pest ortholog of a gene having anucleotide sequence comprising at least one of SEQ ID NOs 11, 31, 134,and 148, or the complement thereof, wherein the two orthologous genesare similar in sequence to such a degree that when the two genes areoptimally aligned and compared, the ortholog has a sequence that is atleast 85% identical to at least one of SEQ ID NOs 11, 31, 134, and 148;or (iii) has a nucleotide sequence encoding an amino acid sequence that,when the two amino acid sequences are optimally aligned and compared, isat least 70% preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99%identical to the amino acid sequence encoded by the nucleotide sequencerepresented by at least one of SEQ ID NOs 11, 31, 134, and 148; or (iv)has a nucleotide sequence encoding an amino acid sequence that, when thetwo amino acid sequences are optimally aligned and compared, is at least70% preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical tothe amino acid sequence represented in any of SEQ ID NOs 216 and
 238. 2.A DNA construct comprising the polynucleotide of claim
 1. 3. The DNAconstruct of claim 2, wherein the DNA construct is an expressionconstruct and wherein the polynucleotide sequence is operably linked toat least one regulatory sequence capable of driving expression of thepolynucleotide sequence.
 4. A host cell comprising the polynucleotide ofclaim
 1. 5. A host cell comprising the DNA construct of claim
 3. 6. Thehost cell of claim 4, wherein the host cell is a prokaryotic or aeukaryotic cell.
 7. The host cell of claim 6, wherein the host cell is abacterial cell or a plant cell.
 8. The host cell of claim 5, wherein thehost cell is a prokaryotic or a eukaryotic cell.
 9. The host cell ofclaim 8, wherein the host cell is a bacterial cell or a plant cell. 10.A composition comprising a dsRNA comprising annealed complementarystrands, one strand of which comprises or consists of a sequence of atleast 21 contiguous nucleotides that is 100% identical to a targetnucleotide sequence within the target gene, wherein said target gene:(i) has a nucleotide sequence comprising at least one of SEQ ID NOs 11,31, 134, and 148, or the complement thereof, or a nucleotide sequencethat, when the two sequences are optimally aligned and compared, is atleast 85% identical to at least one of SEQ ID NOs 11, 31, 134, and 148,or the complement thereof; or (ii) is an insect pest ortholog of a genehaving a nucleotide sequence comprising at least one of SEQ ID NOs 11,31, 134, and 148, or the complement thereof, wherein the two orthologousgenes are similar in sequence to such a degree that when the two genesare optimally aligned and compared, the ortholog has a sequence that isat least 85% identical to at least one of SEQ ID NOs 11, 31, 134, and148; or (iii) has a nucleotide sequence encoding an amino acid sequencethat, when the two amino acid sequences are optimally aligned andcompared, is at least 70% preferably at least 75%, 80%, 85%, 90%, 95%,98% or 99% identical to the amino acid sequence encoded by thenucleotide sequence represented by at least one of SEQ ID NOs 11, 31,134, and 148; or (iv) has a nucleotide sequence encoding an amino acidsequence that, when the two amino acid sequences are optimally alignedand compared, is at least 70% preferably at least 75%, 80%, 85%, 90%,95%, 98% or 99% identical to the amino acid sequence represented in anyof SEQ ID NOs 216 and
 238. 11. The composition of claim 10, furthercomprising at least one suitable carrier, excipient or diluent.
 12. Thecomposition of claim 10, wherein the dsRNA is expressed inside a hostcell.
 13. A method for generating a transgenic plant comprising: (a)transforming a plant cell with the DNA construct of claim 1; (b)regenerating a plant from the transformed plant cell; and (c) growingthe transformed plant under conditions suitable for the expression ofthe double-stranded RNA from the recombinant DNA construct
 14. Atransgenic plant produced by the method of claim
 13. 15. A transgenicplant, reproductive or propagation material for a transgenic plant, or acultured transgenic plant cell, which expresses or is capable ofexpressing at least one dsRNA, wherein said dsRNA comprises annealedcomplementary strands, one strand of which comprises or consists of asequence of at least 21 contiguous nucleotides that 100% identical to atarget nucleotide sequence within the target gene, wherein said targetgene: (i) has a nucleotide sequence comprising at least one of SEQ IDNOs 11, 31, 134, and 148, or the complement thereof, or a nucleotidesequence that, when the two sequences are optimally aligned andcompared, is at least 85% identical to at least one of SEQ ID NOs 11,31, 134, and 148, or the complement thereof; or (ii) is an insect pestortholog of a gene having a nucleotide sequence comprising at least oneof SEQ ID NOs 11, 31, 134, and 148, or the complement thereof, whereinthe two orthologous genes are similar in sequence to such a degree thatwhen the two genes are optimally aligned and compared, the ortholog hasa sequence that is at least 85% identical to at least one of SEQ ID NOs11, 31, 134, and 148; or (iii) has a nucleotide sequence encoding anamino acid sequence that, when the two amino acid sequences areoptimally aligned and compared, is at least 70% preferably at least 75%,80%, 85%, 90%, 95%, 98% or 99% identical to the amino acid sequenceencoded by the nucleotide sequence represented by at least one of SEQ IDNOs 11, 31, 134, and 148; or (iv) has a nucleotide sequence encoding anamino acid sequence that, when the two amino acid sequences areoptimally aligned and compared, is at least 70% preferably at least 75%,80%, 85%, 90%, 95%, 98% or 99% identical to the amino acid sequencerepresented in any of SEQ ID NOs 216 and
 238. 16. A seed produced fromthe transgenic plant of claim
 15. 17. A method to down regulateexpression of a target gene in an insect pest species, comprisingcontacting said insect pest species with an effective amount of at leastone double-stranded ribonucleic acid (dsRNA), wherein said dsRNAcomprises annealed complementary strands, one strand of which comprisesor consists of a sequence of at least 21 contiguous nucleotides that is100% identical to a target nucleotide sequence within the target gene,wherein said target gene: (i) has a nucleotide sequence comprising atleast one of SEQ ID NOs 11, 31, 134, and 148, or the complement thereof,or a nucleotide sequence that, when the two sequences are optimallyaligned and compared, is at least 85% identical to at least one of SEQID NOs 11, 31, 134, and 148, or the complement thereof; or (ii) is aninsect pest ortholog of a gene having a nucleotide sequence comprisingat least one of SEQ ID NOs 11, 31, 134, and 148, or the complementthereof, wherein the two orthologous genes are similar in sequence tosuch a degree that when the two genes are optimally aligned andcompared, the ortholog has a sequence that is at least 85% identical toat least one of SEQ ID NOs 11, 31, 134, and 148; or (iii) has anucleotide sequence encoding an amino acid sequence that, when the twoamino acid sequences are optimally aligned and compared, is at least 70%preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to theamino acid sequence encoded by the nucleotide sequence represented by atleast one of SEQ ID NOs 11, 31, 134, and 148; or (iv) has a nucleotidesequence encoding an amino acid sequence that, when the two amino acidsequences are optimally aligned and compared, is at least 70% preferablyat least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the amino acidsequence represented in any of SEQ ID NOs 216 and
 238. 18. The method ofclaim 17, wherein the insect pest species is a plant pest.
 19. Themethod of claim 18, wherein the plant pest is selected from theLeptinotarsa genus or Lygus genus.
 20. The method of claim 19, whereinthe plant pest is Leptinotarsa decemlineata or Lygus hesperus.